Systemic inhibition of the inflammatory enzyme cyclooxygenase (COX) 2 decreasesthe risk of breast cancer and its recurrence. However, the biology of COX-2 in themulticellular tumor microenvironment is poorly defined.MECKO) comparedto wild type (WT) mice.Mammary tumor onset and multiplicity were examined in ErbB2 transgenic mice thatwere deficient in mammary epithelial cell COX-2  COX-2 in ErbB2-transformed mouse breastcancer cells (COX-2KD), and growth as orthotopic tumors was examined in syngenicrecipient mice, with or without depletion of CD8MECKOmice compared to WT. COX-2MECKO tumors showed decreased expression ofKi67, a proliferation marker, as well as reduced VEGFA, its receptor VEGFR2,endothelial NOS and the vascular endothelial marker CD31, indicating reduced tumorvascularization. COX-2MECKO tumors contained more CD4+ Thelper (Th) cells and CD8+ cytotoxic immune cells (CTL)consistent with increased immune surveillance. The ratio of Th markersTbet (Th1) to GATA3 (Th2) was higher, and levels of Retnla,a M2 macrophage marker, lower, in COX-2MECKO tumor infiltratingleukocytes compared to WT, suggesting a prevalence of pro-immune Th1over immune suppressive Th2 lymphocytes, and reduced macrophagepolarization to the immune suppressive M2 phenotype. Enhanced immune surveillancein COX-2MECKO tumors was coincident with increased intratumoral CXCL9,a T cell chemoattractant, and decreased expression of T lymphocyte co-inhibitoryreceptors CTLA4 and PD-1, as well as PD-L1, the ligand for PD-1. PD-L1 was alsodecreased in IFNγ-treated COX-2KD mouse mammary cancer cells in vitroand, compared to control cells, growth of COX-2KD cells as orthotopic tumorsin immune competent mice was markedly suppressed. However, robust growth ofCOX-2KD tumor cells was evident when recipients were depleted of CD8+cells.Mammary tumor onset was delayed, and multiplicity halved, in COX-2+ immune cell function,possibly through upregulation of immune checkpoints, thereby contributing to tumorimmune escape. COX-2 inhibition may be clinically useful to augment breast cancerimmunotherapy.The data strongly support that, in addition to its angiogenic function, tumor cellCOX-2 suppresses intratumoral cytotoxic CD8 Globat cancer ,4 and hut cancer . PGE2 sieillance .h1)-derivedcytokines that drive antigen-presenting and pro-immune M1 macrophage functions, and bythe direct tumoricidal actions of CD8+ cytotoxic T lymphocytes (CTLs) andnatural killer (NK) cells [h2lymphocyte-derived cytokines polarize macrophages to the M2 phenotype to suppress CTLs,promote angiogenesis and support tumor growth [h2lymphocytes and tumor-associated macrophages (TAM), while Th1 lymphocytes,CTLs and NKs correlate with enhanced survival [2 has emerged as a tumor-derivedmediator that contributes to development of immune tolerance [2 has been linked to immune suppression inhepatocellular carcinoma [2 suppress immunefunction are poorly defined; however, PGE2 suppressed the ability of matureCTLs to kill murine plasmocytoma cells [h1 generation of interferon γ (IFNγ) [In the last decade it has become clear that the tumor microenvironment is critical fortumors to survive and progress. In addition to vascular supply, the interplay of tumorcells with non-malignant cells in the stroma provides growth, survival and motilityadvantages. A central part of the tumor microenvironment is infiltration of immunecells, which can positively or negatively influence tumor progression depending on theirdifferentiation ,8. TumorK) cells . Howeverr growth ,9. In brsurvival , raisingolerance -13. Seveolerance -17, whilarcinoma , lung [1arcinoma , ovarianarcinoma  and breaarcinoma ,15 cancema cells  and inhiγ (IFNγ) ,23, a cyγ (IFNγ) ,24.MECKO) delayed carcinogen-induced mammary tumor onset coincidentwith enhanced markers of anti-tumor type 1 immunity [MECKO mice transgenic for an activated ErbB2 mutant, wedetermined delayed tumor onset and reduced tumor multiplicity, as well as reduced tumorvascularization, compared to wild type (WT). Deletion of COX-2 in tumor cells alsosignificantly impaired maintenance of pro-tumorigenic lymphoid and myeloid cellfunctions thereby facilitating enhanced immune surveillance.We reported that selective deletion of mammary epithelial cell (MEC) COX-2, were backcrossed fully (>9 generations)onto an FVB background and are denoted as wild type (WT) mice. COX-2flox/floxmice were crossed with FVB mice expressing Cre-recombinase under control of themouse mammary tumor virus (mmtv) promoter (Cremmtv), which is used widelyto target transgene expression to MEC. The resulting mice were termedCOX-2MECKO and their characterization is described in our previous work [MECKO were crossed with mice transgenic for theErbB2 (HER2/c-neu) oncogene carrying Eactivating Val664 to Glu664mutation , also expressed under thecontrol of mmtv promoter. Genotype verification was performed by conventional PCRusing primers listed in Table Floxed COX-2 mice, generated by flanking the COX-2 gene between introns 5 and 8 withloxP sites (COX-2ous work . WT and 4Cl) andthen with Trypsin-ethylenediaminetetraacetic acid (EDTA) 0.25% , followed by Dispase and DNase (StemCell Technologies). Thereafter, cellpellets were passed through a 40 μm cell strainer, counted and re-suspendedeither in fluorescence-activated cell sorting (FACS) buffer for flow cytometry(description below) or in degassed MACS buffer (PBS + 0.5% BSA + 2 mM EDTA) forpositive selection of CD45+ cells using CD45-microbeads  according to the manufacturer's instructions.Mice were palpated weekly and considered tumor bearing if a palpable mammary masspersisted for more than one week. On necropsy, tumors were counted and isolated fromsurrounding tissues, after which they were either frozen and stored at -80°C forRNA extraction or fixed in Prefer  overnight andparaffin embedded or digested to obtain single cell suspension for flow cytometry andmicrobead separation. For tissue digestion, tumors were washed with (D)MEM/F12 + 5%fetal bovine serum (FBS) + gentamycin 50 mg/ml, minced and placed in digestion bufferconsisting of 9 parts of wash buffer +1 part collagenase/hyaluronidase . After two hours shaking at 37°C, thesuspensions were centrifuged at 1,000 rpm for five minutes. Pellets were washed andtreated with red cell lysis buffer  to select fortransduced cells. COX-2 knock down in COX-2 shRNA transduced NAF cells (NAF COX-2KD)compared to non-target shRNA transduced cells (NAF nt) was verified by Q-PCR. Cellswere serum starved for 24 hours and then treated with 10 ng/ml IFNγ, with or without 250 nM PGE2 . Fresh IFNγ and PGE2 were added 24 hours later, andcells were harvested (0.25% Trypsin-EDTA) after 48 hour treatment, washed andre-suspended in FACS buffer for flow cytometry analysis.The NAF tumor cell line, which was generated from mammary tumors of ErbB2-transgenicmice, was kindly provided by Dr. Lewis Chodosh (University of Pennsylvania). NAF werecultured in (D)MEM medium containing 10% FBS, 1% L-glutamine and 1%penicillin/streptomycin. For viral transduction, 15,000 cells/well were plated on96-well plates. Mission plKO.1-puro Transduction Lentiviral Particles (20 μl),carrying either non-target control small hairpin RNA (shRNA) or COX-2 shRNA , at 1 × 106cells/well) in LCCM. At 100% confluency, media was replaced with  and 20 ng/ml IFNγ (Peprotech)), or M2 polarizing mix , with or without 250 nM PGE2 . Supernatants were removed 18 hours later and cells lysed for RNAisolation.Bone marrow-derived macrophages (BMDM) were isolated as described . Femurs Total RNA from tumors and cells was isolated ,and reverse transcribed , according to the manufacturer's instructions. Real time quantitative(Q)-PCR of all genes, including 18S ribosomal RNA, was performed using inventoriedgene expression assays and TaqMan Universal PCR Master Mix from Applied Biosystems.PCR products were detected in ABI-PRISM 7900 sequence detection systems (AppliedBiosystems). Results were analyzed using the comparative Ct method, and normalized to18S RNA.Paraffin embedded tumor tissues were sectioned (4 μm). After de-paraffinizationand rehydration, endogenous peroxidase was blocked with 3% hydrogen peroxide. Heatinduced epitope retrieval was performed with 1 mM EDTA . After overnight blocking at 4°C with 5% donkey serum (Sigma) + 0.1% Triton100× (Sigma), sections were incubated with primary antibodies overnight at4°C as follows: anti-Ki67 , anti-CD31 or anti-CXCL9 . Thereafter, the Polink-2 HRP Plus AEC System forImmunohistochemistry  was used,according to the manufacturer's instructions. Slides were then counterstained withhematoxylin , and mounted . Images were taken with a Nikon EclipseE600 microscope using ACT-1 imaging program .6 cell/100 μl/tube. After a five minute incubation with ratanti-mouse CD16/CD32 , 1μg/ml of fluorescein isothiocyanate (FITC) conjugated anti-CD3, PE conjugatedanti-CD4, and AF647 conjugated anti-CD8a, or PE conjugated anti-F4/80 and AF647conjugated anti-CD86 antibodies (Invitrogen) were added. OneComp eBeads  were incubated with anti-CD3 or anti-CD4 or anti-CD8a antibodiesto perform compensation for spectral overlap. NAF COX-2KD and NAF nt (1 ×106 cell/100 μl), were incubated with PE conjugated anti-PD-L1antibody . After a 30-minute incubation, cells werewashed and re-suspended in 500 μl FACS buffer. Unstained tumor cells and cellsincubated with isotype control rat anti-mouse antibody were used as negativecontrols. FACS analysis was performed on a BD FACSCalibur machine . Data was analyzed using FlowJo Research Flow Cytometry AnalysisSoftware .Single cell suspensions from tumor digestions (above) were centrifuged, washed andre-suspended in FACS buffer (PBS + 2% FBS +1 mM EDTA + 0.01% sodium azide), 1 ×106 cells/gland in 100 μl Hanks Balanced Salt Solution) ofnormal WT female mice between 8 to 14 weeks of age. Orthotopic tumor volume wasdetermined weekly using standard caliper measurement. For CD8+ depletion experiments,mice were injected intraperitoneally with 200 μg of an anti-CD8 or isotypecontrol antibody , four days and again two daysprior to injection of tumor cells, and then twice weekly for a further four weeks.Depletion of CD8+ cells was confirmed by flow cytometry of erythrocyte lysed wholeblood , four days and again four weeks after tumorcell injections.NAF COX-2KD and NAF nt tumor cells were injected into the #4 and #9 mammary glands . As appropriate, comparisons were made using logrank analysis, unpairedt-test (with Welch's correction when variances were significantly different byF-test), Mann Whitney test , or, formultiple group comparisons, analysis of variance (ANOVA) followed by Bonferroni'smultiple comparison test.MECKO mice by Q-PCR and Westernblotting, and loss of PGE2 generation by COX-2MECKO cells wasestablished by mass spectrometry [2 production were unchanged inperipheral macrophages isolated from COX-2MECKO compared to WT confirmingthe selectivity of the deletion [MECKO mice compared to their WT littermates  NOS, the angiogenic factor VEGFA and its receptor VEGFR2Figure , in COX-s Figure . Immunoss Figure .MECKO tumors were analyzed by flow cytometry and Q-PCR tocompare the populations of infiltrating immune cells and their phenotypes. By flowcytometry, there was no difference in the total number of F4/80+ TAMsbetween WT and COX-2MECKO tumors  cells, as well asCD3+CD8+ CTLs and CD3-CD8+ cells,encompassing NK and dendritic cells /GATA3 (Th2 marker) tended to behigher in COX-2MECKO tumors compared to WT . Gene expression of FoxP3, a marker for Treg,was not altered and there was no difference in mRNA for macrophage type 1 cytokinesTNFα and IFNγ or an M1 macrophage marker CD86, in CD45+ TILs,suggesting no major change in M1 polarization in this disease model. COX-2-derivedPGE2 has been implicated in driving the immune suppressive phenotypetypically associated with TAM [2 treatment significantly increased theexpression of M2 marker Arginase 1, a key enzyme in suppression of T cell function,in both M1 and M2 polarized bone-marrow derived macrophages , suggesting thattumor cell PD-L1 was suppressed by COX-2 deficiency. Indeed, NAF COX-2KD, which,compared to NAF nt, grew poorly as orthotopic tumors in immune competent syngenicmice  or in isolated CD45+ TILs, compared to WT. It is likely thatthe relevance of COX-2-mediated paracrine control of M1 macrophage function to tumorprogression varies between models. Retnla , acytokine derived from alternatively activated M2 type macrophages [+ TILs from COX-2MECKOtumors suggesting reduced M2 polarization, a possible reflection of reducedTh2-derived cytokines in the COX-2MECKO microenvironment and/orloss of paracrine COX-2-derived PGE2 activity, which augments M2 polarizationof BMDM in vitro.TAM are abundant in mammary tumors and their density is generally directly correlatedwith disease severity and prognosis ,43. Simiectively . We repoy tumors . In the MECKO tumors. It may be that there is simply an increased immune cellrecruitment to breast tumors lacking epithelial COX-2. Indeed, we saw a dramaticincrease in tumor cell expression of the T cell chemokine CXCL9 in COX-2MECKOtumors, consistent with a recent report in patients with invasive breast cancer thattumor cells are the major source of CXCL9 [2 suppressed IFNγ-induced CXCL9 levelsin MCF-7 and MDA-MB 231 breast cancer cells, and COX inhibitors increased CXCL9secretion. Despite the higher CXCL9 levels in COX-2MECKO tumors, however, theabsolute number of CD3+ cells by flow cytometry was not higher than in WTtumors suggesting a local influence of tumor cell COX-2 derived mediators in limitingimmune cell function rather than a simple recruitment effect.As a whole, our analysis of the tumor microenvironment strongly supported a shifttowards enhanced helper and effector T lymphocyte recruitment and function inCOX-2of CXCL9 . In the MECKO tumors. The PD-1-PD-L1 interaction is of particular interest inthis regard since PD-1 expression in tissues is induced by inflammatory signals where itacts to suppress T cell activity and limit collateral tissue damage [2 to restore PD-L1 expression levels in NAF COX-2KD may suggestdistinct actions of autocrine and paracrine PGE2, or indicate a role forother COX-2-derived products, in tumor cell COX-2 mediated control of PD-1 expression.The pathways through which COX-2-derived PGE2/other prostanoids control tumorcell expression of PD-L1 and other immune modulators are currently underinvestigation.Intense interest in cancer immunotherapy has focused recently on immune checkpoints,whose function to dampen immune responses is important for self tolerance and control ofphysiological immune responses. Two central and well-studied immune checkpoints are theco-inhibitory receptors CTLA4 and PD-1; antagonists to both are currently in clinicaltrials for melanoma and other cancers . Engageme damage . We reas+ tumor infiltrationbolstered chemotherapeutic responses in human breast cancer and mouse models [+ cytotoxic immune cell infiltration and function mayopen new avenues to develop targeted strategies for COX-2 inhibition in combination withcytotoxic drugs. Further, there have been significant advances in cancer immunotherapyusing antibodies to block CTLA4 or PD-1 co-inhibitory function, thereby augmentinganti-tumor immunity [Our study provides significant insight into the complex autocrine and paracrinefunctions of mammary epithelial COX-2 in ErbB2-induced breast cancer and suggests thattumor cell COX-2 is an important component in establishing a permissive immunemicroenvironment. Recent studies indicated that CD8immunity ,47-49. Timmunity  induced immunity . We specThe data strongly support that, in addition to its angiogenic function, tumor cellCOX-2-derived mediators suppress anti-tumor immune cell function, possibly throughupregulation of inhibitory immune checkpoints, contributing to tumor immune escape.COX-2 inhibition may be clinically useful to augment breast cancer immunotherapy.BMDM: bone marrow derived macrophages; BSA: bovine serum albumin; COX: cycloogygenase;CTL: cytotoxic T lymphocyte; CTLA-4: cytotoxic T lymphocyte antigen 4; (D)MEM:(Dulbecco's) modified Eagle's medium; EDTA: ethylenediaminetetraacetic acid; FACS:fluorescence-activated cell sorting; FBS: fetal bovine serum; IFN: interferon; KO: knockout; KD: knock down; MEC: mammary epithelial cell; mmtv: mouse mammary tumor virus; NK:natural killer cells; nt: non-target; PCR: polymerase chain reaction; PD-1: programeddeath 1; PD-L1: programmed death ligand 1; PG: prostaglandin; shRNA: small hairpin RNA;TAM: tumor associated macrophage; TIL: tumor infiltrating leukocyte; TNF-α: tumornecrosis factor-α; WT: wild type.The authors declare that they have no competing interests.NM made substantial contributions to the study design, data acquisition, analysis andinterpretation, as well as the writing and editing, of the manuscript. EPC contributedto data acquisition and analysis in the macrophage and flow cytometry experiments. RAEcontributed to the design of the immune depletion experiments. VN contributed to thedata acquisition. RHV contributed to the analysis and interpretation of the immunefunction data. EMS directed the conception and design of the study and the experimentalwork, contributed to the analysis and interpretation of data and critically revised themanuscript for important intellectual content. All authors read and approved the finalmanuscript.


In chronic cardiac hypertrophy, however, ANP levels are markedly increased and GC-A/cGMP responses to ANP are blunted due to receptor desensitization.Cardiac atrial natriuretic peptide (ANP) regulates arterial blood pressure, moderates cardiomyocyte growth, and stimulates angiogenesis and metabolism. ANP binds to the transmembrane guanylyl cyclase (GC) receptor, GC-A to exert its diverse functions. This involves a cGMP-dependent signaling pathway preventing pathological [Ca2+ levels ([Ca2+i]). This pathway involves the activation of TRPC3/C6 Ca2+ channels  by GC-A which forms a stable complex with TRPC3/C6 channels. Our results indicate that the resulting TRPC3/C6-mediated Ca2+ entry then stimulates Calmodulin Kinase II (CaMKII) to phosphorylate L-type Ca2+ channels leading to increased L-type Ca2+ channel mediated Ca2+ current and a rise in intracellular Ca2+ levels.Here we show that in this situation ANP binding to GC-A stimulates a novel cGMP-independent signaling pathway in cardiac myocytes, resulting in pathologically elevated intracellular Ca2+i-homeostasis. Under physiological conditions, activation of a cGMP-dependent pathway moderates the Ca2+i-enhancing action of hypertrophic factors such as Angiotensin II. By contrast, a cGMP-independent pathway predominates under pathophysiological conditions, when GC-A is desensitized by high ANP levels. The concomitant rise in [Ca2+i] is likely to increase the propensity to cardiac hypertrophy and arrhythmias.These observations reveal a dual role of the ANP/GC-A signaling pathway in the regulation of cardiac myocyte Ca


Arabidopsis thaliana AtU11/U12-31K protein has been shown to be indispensible for proper U12 intron splicing and for normal growth and development of Arabidopsis plants. Here, we assessed the functional roles of the rice (Oryza sativa) OsU11/U12-31K protein in U12 intron splicing and development of plants. The U11/U12-31K transcripts were abundantly expressed in the shoot apical meristems (SAMs) of Arabidopsis and rice. Ectopic expression of OsU11/U12-31K in AtU11/U12-31K-defecient Arabidopsis mutant complemented the incorrect U12 intron splicing and abnormal development phenotypes of the Arabidopsis mutant plants. Impaired cell division activity in the SAMs and inflorescence stems observed in the AtU11/U12-31K-deficient mutant was completely recovered to normal by the expression of OsU11/U12-31K. Similar to Arabidopsis AtU11/U12-31K, rice OsU11/U12-31K was determined to harbor RNA chaperone activity. Collectively, the present findings provide evidence for the emerging idea that the U11/U12-31K protein is an indispensible RNA chaperone that functions in U12 intron splicing and is necessary for normal development of monocotyledonous plants as well as dicotyledonous plants.U12 intron-specific spliceosomes contain U11 and U12 small nuclear ribonucleoproteins and mediate the removal of U12 introns from precursor-mRNAs. Among the several proteins unique to the U12-type spliceosomes, an  Drosophila, zebra fish, and human have been demonstrated Splicing of both the major class of U2-dependent introns and minor class of U12-dependent introns is an indispensible step in the regulation of gene expression in eukaryotes. Although U12-type minor introns, which constitute less than 0.5% of all introns in animals and plants Splicing of the minor class of U12-type introns and major class of U2-type introns is catalyzed by the minor spliceosome and major spliceosome, respectively Arabidopsis AtU11/U12-35K was shown to facilitate recognition of the 5′ splice site Arabidopsis AtU11/U12-31K was essential for U12-type intron splicing by functioning as an RNA chaperone, and the AtU11/U12-31K-dependent U12 splicing was shown to be critical for normal plant development Oryza sativa) OsU11/U12-31K protein in U12 intron splicing and the development of plants. By functional complementation of OsU11/U12-31K in the AtU11/U12-31K-defecient Arabidopsis mutant plants, we provide evidence demonstrating that U11/U12-31K proteins are functionally conserved RNA chaperones in Arabidopsis thaliana and rice, which are crucial for correct U12 intron splicing and normal development of monocotyledonous plants as well as dicotyledonous plants.Despite the well-conserved sequences of the minor spliceosome-associated proteins in animals and plants, the significance and biological roles of many minor spliceosomal proteins have not yet been experimentally demonstrated. It has been determined that U11/U12-65K, U11/U12-59K, and U11/U12-48K proteins are integral proteins in animals necessary for 5′ splice site selection and branchpoint site selection AtU11/U12-31K was determined to be ubiquitously expressed in all Arabidopsis organs, including rosette leaves, cauline leaves, inflorescence stem, floral buds, and flowers U11/U12-31K gene in Arabidopsis and rice by in situ hybridization analysis. Results showed that U11/U12-31K was abundantly expressed in the shoot apical meristem (SAM) of Arabidopsis and rice  . SeveralArabidopsis AtU11/U12-31K was shown to harbor RNA chaperone activity E. coli BX04 mutant cells, which lack four cold shock protein (CSP) genes that are bacterial RNA chaperones S-transferase (GST)-31K protein and GST-CspA protein were purified from E. coli  and utilized for nucleic acid-melting analysis. The DNA-melting ability of OsU11/U12-31K was evaluated by using a partially double-stranded DNA molecule that produces fluorescence signals upon melting. Strong fluorescent signals were observed upon the addition of recombinant GST-31K or GST-CspA (positive control), but not the addition of GST protein (negative control), to the reaction mixture (in vitro DNA-melting ability. We next performed the RNase T1 cleavage assay to determine whether OsU11/U12-31K is capable of melting RNA secondary structures. The addition of RNase T1 to the RNA substrates transcribed from the pET-22b(+) plasmid produced several RNase T1-resistant bands , all of which are closely related to cell division, seed maturation, and seedling growth Arabidopsis AtU11/U12-31K mutants were completely recovered in the complementation lines expressing rice OsU11/U12-31K . Plants were also grown in half-strength Murashige and Skoog (MS) medium containing 1% sucrose. The AtU11/U12-31K knockdown Arabidopsis mutant plants (amiR1-4), in which the expression of AtU11/U12-31K is downregulated by an artificial microRNA-mediated knockdown strategy , were described in our previous report OsU11/U12-31K gene under the control of the cauliflower mosaic virus 35S promoter was introduced into the Arabidopsis amiR1-4 mutant by vacuum infiltration Agrobacterium tumefaciens GV3101. The T3 homozygous lines were selected and used for phenotype analysis.U11/U12-31K in different organs and cells, in situ hybridization analysis was conducted using different tissues of Arabidopsis and rice. Plant samples were fixed with 4% paraformaldehyde in 50 mM sodium phosphate buffer (pH 7.0), embedded in paraffin, and sliced into thin sections. The RNA probes were synthesized in vitro using a digoxigenin RNA labeling kit . The probes were hybridized to the samples, and the signals were detected by chemiluminescence. For scanning electron microscopy (SEM) observation, the samples were fixed with a mixture of 2% glutaraldehyde and 2% paraformaldehyde, and were post-fixed with 1% osmium tetroxide in 50 mM cacodylate buffer (pH 7.2). After dehydrating the samples with a series of alcohols, the samples were dried with a HCP-2 critical point dryer , coated with gold in a Emitec K550 ion sputter, and observed with a Hitachi S-2400 scanning electron microscope . For light microscope observation, the samples which were embedded in LR White resin were sectioned into thin sections, stained with 0.1% toluidine blue, and examined with a light microscope .To determine the expression patterns of Arabidopsis mutant (amiR1-4), and OsU11/U12-31K-expressing amiR1-4 plants. Five to ten micrograms of total RNAs were treated with RQ1 DNase  and further purified using an RNeasy clean-up kit . RT-PCR analysis of the splicing patterns was conducted as described previously For the analysis of splicing patterns of U12 intron-containing genes, total RNAs were extracted from 20-day-old wild-type, AtU11/U12-31K knockdown E. coli BX04 mutant cells, OsU11/U12-31K cDNA was cloned into the pINIII vector. The cold shock test for E. coli was conducted as described previously E. coli BX04 mutant cells, and the growth of cells was monitored in Luria-Bertani (LB) medium at low temperatures. In the nucleic acid-melting assay, the 78-nucleotide-long hairpin-shaped DNA molecule labeled with a fluorophore (tetramethyl rhodamine) and quencher (dabcyl) was synthesized as described previously 32P-labeled RNA substrates were prepared by transcription of the pET-22b(+) plasmid using T7 RNA polymerse (Promega). The RNA substrates were incubated with recombinant GST-31K fusion proteins, and the reaction products were separated on an 8% acrylamide gel. All experimental conditions were maintained as described previously In the cold shock assay using the Figure S1Morphology of inflorescence stems of the plants. (A) Light micrographs of floral bud regions of 7-week-old wild-type (WT), knockdown mutant (amiR1-4), and complementation line expressing OsU11/U12-31K gene (amiR1-4/Os31K). Scale bar  = 1mm. (B) SEM of inflorescence stems of 7-week-old wild-type, mutant, and complementation line.(TIF)Click here for additional data file.Figure S2Abnormal splicing patterns of U12-type introns in the amiR1-4 mutant and complementation lines. The splicing patterns of several U12 intron-containing transcripts were analyzed by RT-PCR in wild-type (WT), knockdown plants (amiR1-4), and complementation lines expressing OsU11/U12-31K gene (amiR1-4/Os31K). The experiment was repeated three times using different batches of RNA samples, and similar results were obtained. The gray boxes with numbers represent exons, and the dashed and solid lines represent U12 and U2 introns, respectively.(TIF)Click here for additional data file.Figure S3Purification of GST-31K fusion proteins. The recombinant GST fusion proteins were purified in E. coli and the purified GST, GST-CspA and GST-Os31K fusion proteins were analyzed by SDS-PAGE.(TIF)Click here for additional data file.Table S1List of U12 intron-containing genes investigated in this study and their splicing patterns in the amiR1-4 mutant plant.(RTF)Click here for additional data file.


The innate immune system is essential for controlling viral infections, but several viruses have evolved strategies to escape innate immunity. RIG-I is a cytoplasmic viral RNA sensor that triggers the signal to induce type I interferon production in response to viral infection. RIG-I activation is regulated by the K63-linked polyubiquitin chain mediated by Riplet and TRIM25 ubiquitin ligases. TRIM25 is required for RIG-I oligomerization and interaction with the IPS-1 adaptor molecule. A knockout study revealed that Riplet was essential for RIG-I activation. However the molecular mechanism underlying RIG-I activation by Riplet remains unclear, and the functional differences between Riplet and TRIM25 are also unknown. A genetic study and a pull-down assay indicated that Riplet was dispensable for RIG-I RNA binding activity but required for TRIM25 to activate RIG-I. Mutational analysis demonstrated that Lys-788 within the RIG-I repressor domain was critical for Riplet-mediated K63-linked polyubiquitination and that Riplet was required for the release of RIG-I autorepression of its N-terminal CARDs, which leads to the association of RIG-I with TRIM25 ubiquitin ligase and TBK1 protein kinase. Our data indicate that Riplet is a prerequisite for TRIM25 to activate RIG-I signaling. We investigated the biological importance of this mechanism in human cells and found that hepatitis C virus (HCV) abrogated this mechanism. Interestingly, HCV NS3-4A proteases targeted the Riplet protein and abrogated endogenous RIG-I polyubiquitination and association with TRIM25 and TBK1, emphasizing the biological importance of this mechanism in human antiviral innate immunity. In conclusion, our results establish that Riplet-mediated K63-linked polyubiquitination released RIG-I RD autorepression, which allowed the access of positive factors to the RIG-I protein. The cytoplasmic viral RNA sensor RIG-I recognizes various types of pathogenic viruses and evokes innate immune responses, whereas several viruses have evolved strategies to escape the host innate immune responses. RIG-I triggers a signal to induce type I interferon and inflammatory cytokines. RIG-I activation is regulated by K63-linked polyubiquitin chains mediated by the ubiquitin ligases TRIM25 and Riplet; however, the functional difference between the two ubiquitin ligases remains unclear, and the molecular mechanism underlying Riplet-mediated RIG-I activation is unknown. We revealed sequential roles of the two ubiquitin ligases during RIG-I activation and found that Riplet-mediated polyubiquitination of the RIG-I repressor domain released RIG-I autorepression of its N-terminal CARDs responsible for triggering the signal, which resulted in an association with TRIM25 ubiquitin ligase and TBK1 protein kinase. Interestingly, we found that this mechanism was targeted by hepatitis C virus, which is a major cause of hepatocellular carcinoma. This result emphasizes the vital role of Riplet-mediated release of RIG-I RD autorepression in antiviral responses. Our results establish that Riplet releases RIG-I RD autorepression and demonstrated the biological significance of this mechanism in human innate immune responses. The innate immune system is essential for controlling virus infections, and several viruses have evolved strategies to evade host innate immune responses. Cytoplasmic viral RNA is recognized by RIG-I-like receptors, including RIG-I and MDA5 Several ubiquitin ligases are involved in regulating the RIG-I-dependent pathway, and RIG-I itself is regulated by ubiquitin chains Another E3 ubiquitin ligase, Riplet, binds RIG-I RD and mediates the K63-linked polyubiquitination of RIG-I RD in vitro and in vivoHepatitis C virus (HCV) is a major cause of hepatocellular carcinoma (HCC) worldwide. HCV RNA is primarily recognized by RIG-I In mouse embryonic fibroblasts (MEFs), TRIM25 is essential for type I IFN production in response to SeV infection We performed reporter gene assays with p125luc (IFN-β reporter) using either RIG-I CARDs fragment or full-length RIG-I expression vectors. It is known that ectopic expression of RIG-I CARDs fragment activates the signaling even in the absence of stimulation with RIG-I ligand We then investigated whether the two ubiquitin ligases associate with RIG-I after stimulation. First, we performed immunoprecipitation assay and found that endogenous Riplet and TRIM25 bound to endogenous RIG-I after stimulation with HCV dsRNA . Second,ΔCTD) abrogated Riplet effect on RIG-I signaling  activity. Thus, Riplet disruption by NS3-4A might be a cause of liver disease induced by HCV infection.All animal studies were carried out in strict accordance with Guidelines for Animal Experimentation of the Japanese Associations for Laboratory Animal Science. The protocols were approved by the Animal Care and Use Committee of Hokkaido University, Japan (Permit Number: 08-0245 and 09-0215).HEK293, Vero, and HepG2 cells were cultured in Dulbecco's modified Eagle's medium low glucose medium (D-MEM) with 10% heat-inactivated fetal calf serum (FCS) (Invitrogen). HeLa cells were cultured in minimum Eagle's medium with 2 mM L-glutamine and 10% heat-inactivated FCS. HEK293FT cells were maintained in D-MEM high glucose medium containing 10% of heat-inactivated FCS (Invitrogen). Human hepatocyte cell line with HCV 1b full-length replicons (O cells) and O curred cells (Oc cells) were kindly gifted from Kato N VSV Indiana strain and SeV HVJ strain were amplified using Vero cells. To determine the virus titer, we performed plaque assay using Vero cells. HCV JFH1 was amplified using HuH7.5 cells.Generation of IPS-1 KO and Riplet KO mice were described previously ΔRD (RIG-I-dRD), FLAG-tagged RIG-I RD, C-terminal HA-tagged TRIM25, C-terminal HA-tagged Riplet, and Riplet-ΔRING (Riplet-DN) plasmids were described previously TTC CCG TGT GGC TGG CCG CGG CCG CCC TCG GCT GCA TCA TCT GCC, and Riplet-3A-R: GGC AGA TGA TGC AGC CGA GGG CGG CCG CGG CCA GCC ACA CGG GAA. RIG-I K172R and RIG-I K788R expression vectors was constructed by PCR-mediated mutagenesis using primers, RIG-I K172R-F, RIG-I K172R-R, RIG-I K788R-F and RIG-I-K788R-R, and pEF-BOS/FLAG-RIG-I plasmid as a template. The primer sequences are RIG-I K172R-F: GGA AAA CTG GCC CAA AAC TTT GAG ACT TGC TTT GGA GAA AG, RIG-I K172R-R: CTT TCT CCA AAG CAA GTC TCA AAG TTT TGG GCC AGT TTT CC, RIG-I-K788R-F: TGC ATA TAC AGA CTC ATG AAA GAT TCA TCA GAG ATA GTC AAG AA, and RIG-I-K788-R: CTT GAC TAT CTC TGA TGA ATC TTT CAT GAG TCT GTA TAT GCA G. RIG-I 5KR expression vectors were constructed by PCR-mediated mutagenesis using primers, RIG-I 849 851 RR-F, RIG-I 849 851 RR-R, RIG-I 888R-F, RIG-I 888R-R, RIG-I 907 909 RR-F, RIG-I 907 909 RR-R, and pEF-BOS/FLAG-RIG-I plasmid as a template. The primer sequences are RIG-I 849 851 RR-F: AGT AGA CCA CAT CCC AGG CCA AGG CAG TTT TCA AGT TTT G, RIG-I 849 851 RR-R: CAA AAC TTG AAA ACT GCC TTG GCC TGG GAT GTG GTC TAC T, RIG-I 888R-F: GAC ATT TGA GAT TCC AGT TAT AAG AAT TGA AAG TTT TGT GGT GGA GG, RIG-I 888R: CCT CCA CCA CAA AAC TTT CAA TTC TTA TAA CTG GAA TCT CAA ATG TC, RIG-I 907 909RR-F: GTT CAG ACA CTG TAC TCG AGG TGG AGG GAC TTT CAT TTT GAG AAG, RIG-I 907 909RR-R: CTT CTC AAA ATG AAA GTC CCT CCA CCT CGA GTA CAG TGT CTG AAC. HCV cDNA fragment encoding NS3-4A of JFH1 strain was cloned into pCDNA3.1 (-) vector. The mutation on catalytic site of NS3-4A S139A was constructed by PCR-mediated mutagenesis using primers, NS3-4A S139A-F and NS3-4A S139A-R, and pCDNA3.1 (-)/NS3-4A plasmid as a template. The primer sequences are NS3-4A S139A-F: TTC GAC CTT GAA GGG GTC CGC GGG GGG ACC GGT GCT TTG C and NS3-4A S139A-R: AAG CAC CGG TCC CCC CGC GGA CCC CTT CAA GGT CGA AAG G.Expression vectors encoding for N-terminal FLAG-tagged RIG-I, N-terminal FLAG-tagged RIG-I CARDs (dRIG-I), FLAG-tagged RIG-I Total RNA was extracted with TRIZOL (Invitrogen), after which the samples were treated with DNaseI to remove DNA contamination. Reverse transcription was performed using High Capacity cDNA Reverse Transcription Kit (ABI). Quantitative PCR analysis was performed using Step One software ve2.0. (ABI) with SYBER Green Master Mix (ABI). HCV ss and dsRNA was in vitro synthesized with SP6 and/or T7 RNA polymerase using 3′ UTR of HCV cDNA as template as described previously Cells were plated onto microscope cover glasses (matsunami) in a 24-well plate. The cells were fixed for 30 min using 3% formaldehyde in PBS and permeabilized with 0.2% Triton X-100 for 15 min. Fixed cells were blocked with 1% bovine serum albumin in PBS for 10 min and labeled with the indicated primary Abs for 60 min at room temperature. Alexa-conjugated secondary Abs were incubated for 30 min at room temperature to visualize staining of the primary Ab staining. Samples were mounted on glass slides using Prolong Gold (Invitrogen). Cells were visualized at a magnification of ×63 with an LSM510 META microscope (Zeiss). Data collected with confocal microscopy were analyzed with ZEISS LSM Image Examiner software. NS3, RIG-I, TBK1, IPS-1, and p-TBK1 were stained with anti-NS3 goat pAb (abcam), anti-RIG-I mouse mAb , anti-NAK (TBK1) rabbit mAb , anti-MAVS (IPS-1) rabbit pAb (Bethyl Laboratories Inc), and anti-p-TBK1 rabbit mAb ,Renilla luciferase. The total amounts of plasmids were normalized using an empty vector. Cells were lysed in a lysis buffer (Promega), and luciferase and Renilla luciferase activities were determined using a dual luciferase assay kit (Promega). Relative luciferase activities were calculated by normalizing the luciferase activity by control. HCV dsRNA (3′ UTR polyU/UC region) was synthesized using T7 and SP6 RNA polymerase as described previously HEK293 cells were transiently transfected in 24-well plates using FuGene HD (Promega) or lipofectamine 2000 (Invitrogen) with expression vectors, reporter plasmids (IFN-β: p125luc), and an internal control plasmid coding AAA CUG AAA GGG AGA AGU GAA AGU G; and (antisense strand) UCAC UUU CAC UUC UCC CUU UCA GUU . Biotin was conjugated at the U residue at the 3′-end of the antisense strand (underlined). Biotinylated dsRNA was phosphorylated by T4 polynucleotide kinase (TAKARA). dsRNA was incubated for one hour at 25°C with 10 µg of protein from the cytoplasmic fraction of cells that were transfected with Flag-tagged RIG-I, Riplet, and/or HA-tagged ubiquitin expressing vectors. This mixture was added into 400 µl of lysis buffer  containing 25 µl of streptavidine Sepharose beads, rocked at 4°C for two hours, harvested by centrifugation, washed three times with lysis buffer, and resuspended in SDS sample buffer.RNA used for the assay was purchased from JBioS. The RNA sequences are as follows: (sense strand) 7) were infected with or without VSV at MOI = 10 for eight hours, after which cell extracts were prepared with lysis buffer . Immunoprecipitation used an anti-RIG-I Rabbit monoclonal antibody . To detect endogenous K63-linked polyubiquitin chain that is ligated to RIG-I, 6×107 of mouse splenocyte were infected with SeV at MOI = 0.2 for 24 hours. Immunoprecipitation was performed with anti-RIG-I mAb (D14G6). Anti-K63-linkage specific polyubiquitin (D7A11) Rabbit mAb  was used for western blotting. HEK293FT cells were transfected with or without 0.8 µg of HCV dsRNA in a 6-well plate. HCV dsRNA (HCV 3′ UTR polyU/UC region) was synthesized using T7 and SP6 RNA polymerase as previously described 3VO4, 20 mM iodoacetamide, and 2 mM phenylmethylsulfonyl fluoride) to dissociate unanchored polyubiquitin chain Splenocytes (1×10GGA ACA UCU UGU AGA CAU Utt and (anti-sense) AAU GUC UAC AAG AUG UUC CCac. siRNA was transfected into cells using RNAiMax Reagent (Invitrogen) according to the manufacture's instructions.siRNAs for human Riplet  and negative control were purchased from Ambion. siRNA sequences for Riplet are: (sense) FLAG-tagged Riplet was expressed in HEK293FT cells, and cell lysate was prepared with the lysis buffer described above. The protein was immunoprecipitated with anti-FLAG antibody and protein G sepharose beads, and washed with Buffer B . The samples were suspended in 50 µl of Buffer B, and incubated with 400 ng of recombinant NS3-4A (rNS3-4A) protein at 37°C for one hour, and then subjected to SDS-PAGE analysis. The NS3-4A protein was purchased from AnaSpec Inc (CA). N-terminal GST-fused Riplet (1–210 aa) (rRiplet) was purchased from Abnova. 500 ng of rRiplet was incubated with or without 500 ng of rNS3-4A in 10 µl of reaction buffer (20 mM Tris-HCl (7.5), 4% Glycerol, 5 mM DTT, 150 mM NaCl, 0.1% of Triton-X100, 0.9% polyvinyl alcohol) at 37°C for 30 min.The accession numbers are Riplet (BAG84604), TRIM25 (NP_005073), TBK1 (NP_037386), IKK-ε (AAF45307), IPS-1 (BAE79738), RIG-I (NP_055129), and G3BP (CAG38772).Figure S1K63-linked polyubiquitination of RIG-I RD. HA-tagged ubiquitin and FLAG-tagged RIG-I RD expression vectors were transfected into HEK293FT cells. 24 hours after transfection, the cells were infected with VSV at MOI = 1 for six hours. Then, cell lysate was prepared. Immunoprecipitation was carried out using anti-FLAG antibody. The samples were subjected to SDS-PAGE, and the proteins were detected by western blotting using anti-HA, FLAG, and K63-linked polyubiquitin specific antibodies.(TIF)Click here for additional data file.Figure S2Intracellular localization of RIG-I, NEMO, and p-TBK1 proteins. (A) HeLa cells were transfected with HCV dsRNA using lipofectamine 2000 reagent. The cells were fixed six hours after transfection. The microscopic analysis was performed using anti-RIG-I mAb (Alme-1) and anti-NEMO pAb. (B) HeLa cells were transfected with HCV dsRNA using lipofectamine 2000 reagent (Invitrogen). The cells were fixed at indicated hour. The microscopic analysis was performed using anti-RIG-I mAb (Alme-1). (C) HepG2 cells were transfected with HCV dsRNA using lipofectamine 200 reagent. The cells were fixed six hours after the transfection. The microscopic analysis was performed using anti-RIG-I (Alme-1) mAb and anti-p-TBK1 mAb.(TIF)Click here for additional data file.Figure S3NS3-4A of HCV cleaves IPS-1 and Riplet but not IKK-ε. (A) HA-tagged Riplet was transfected into HEK293 cells together with NS3-4A. 24 hours after transfection, cell lysate was prepared and subjected to SDS-PAGE. The proteins were detected by western blotting and CBB staining.  HA-tagged IKK-ε (B) or IPS-1 (C) expression vectors were transfected into HEK293FT cells with or without NS3-4A of HCV expression vector. 24 hours after the transfection, the cell lysate was prepared, and analyzed by SDS-PAGE. The proteins were detected by western blotting using anti-HA or anti-β actin antibodies. (D) HA-tagged IPS-1 or HA-tagged Riplet expression vector was transfected into HEK293FT cells with or without NS3-4A expression vectors. 24 hours after transfection, cell lysate was prepared and subjected to SDS-PAGE. The proteins were detected by western blotting using anti-HA antibody.  N-terminal FLAG-tagged Riplet (E) or C-terminal HA-tagged Riplet (F) expression vector was transfected into HEK293FT cells with NS3-4A or NS3-4A*. 24 hours after the transfection, cell lysates were analyzed by SDS-PAGE. (G) HA-tagged wild-type Riplet or mutant Riplet-C21A expression vector were transfected into HEK293FT cells with NS3-4A or NS3-4A*. 24 hours after the transfection, the cell lysate was prepared, and analyzed by SDS-PAGE. The proteins were detected by western blotting using anti-HA or anti-β actin antibodies.  RIG-I, Riplet, Riplet-3A (H), and/or Riplet C21A (I) mutant expression vectors were transfected into HEK293 cells together with p125luc reporter and Renilla luciferase. 24 hours after transfection, luciferase activity was measured.(TIF)Click here for additional data file.Figure S4siRNA for Riplet or control was transfected into HeLa cells in 24-well plate using RNAi MAX (Invitrogen) according to manufacture's protocol. 48 hours after transfection, the cells were transfected with 100 ng of HCV dsRNA. Six hours after transfection, the cells were fixed and stained with anti-RIG-I mAb (Alme-1) and anti-mouse Alexa-488 Ab.(TIF)Click here for additional data file.


In Table 2, the first column, seventh row should read "PRE-TRIAL Guidelines (72.2%)".


Autotaxin (ATX) is a secreted glycoprotein with the lysophospholipase D (lysoPLD) activity to convert lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), a bioactive lysophospholipid involved in diverse biological actions. ATX is highly expressed in some cancer cells and contributes to their tumorigenesis, invasion, and metastases, while in other cancer cells ATX is silenced or expressed at low level. The mechanism of ATX expression regulation in cancer cells remains largely unknown.In the present study, we demonstrated that trichostatin A (TSA), a well-known HDAC inhibitor (HDACi), significantly induced ATX expression in SW480 and several other cancer cells with low or undetectable endogenous ATX expression. ATX induction could be observed when HDAC3 and HDAC7 were down-regulated by their siRNAs. It was found that HDAC7 expression levels were low in the cancer cells with high endogenous ATX expression. Exogenous over-expression of HDAC7 inhibited ATX expression in these cells in a HDAC3-dependent manner. These data indicate that HDAC3 and HDAC7 collaboratively suppress ATX expression in cancer cells, and suggest that TSA induce ATX expression by inhibiting HDAC3 and HDAC7. The biological significance of this regulation mechanism was revealed by demonstrating that TSA-induced ATX protected cancer cells against TSA-induced apoptosis by producing LPA through its lysoPLD activity, which could be reversed by BrP-LPA and S32826, the inhibitors of the ATX-LPA axis.We have demonstrated that ATX expression is repressed by HDAC3 and HDAC7 in cancer cells. During TSA treatment, ATX is induced due to the HDAC3 and HDAC7 inhibition and functionally antagonizes the TSA-induced apoptosis. These results reveal an internal HDACi-resistant mechanism in cancer cells, and suggest that the inhibition of ATX-LPA axis would be helpful to improve the efficacy of HDACi-based therapeutics against cancer. Differe) family . The bro) family .ATX plays roles in the immune  and the ATX expression is inducible by VEGF, EGF, bFGF and BMP-2, but inhibited by TGF-β and several cytokines, including IL-1, IL-4 and IFN-γ . We havein vitro and in vivo in transformed cells, but not in normal cells [Histone deacetylases (HDACs) comprise of a family of 18 genes, which are grouped into classes I-IV based on their homology to their respective yeast orthologues . In addial cells . More thal cells . Howeveral cells .In this study, we demonstrated that TSA, a well-known HDACi, induced ATX expression in various cancer cell lines. HDAC3 and HADC7, as the targets of TSA, were involved in ATX expression regulation in cancer cells. The TSA-induced ATX protected cancer cells from TSA-induced apoptosis by producing LPA through its lysoPLD activity, while BrP-LPA and S32826, the inhibitors of ATX-LPA axis, promoted the TSA-induced apoptosis. These results suggest that inhibition of ATX-LPA axis would potentially improve the efficacy of HDAC inhibitors in cancer treatment.To investigate whether ATX expression is epigenetically regulated in cancer cells, we tested the roles of DNA methylation and protein acetylation in ATX expression in a colon cancer cell line SW480, where endogenous ATX expression was not detectable. HDAC inhibitor trichostatin A (TSA), but not 5-aza-2'-deoxycytidine, an inhibitor of DNA methylation, induced ATX expression in SW480 cells Figure . This inA panel of different cancer cell lines was used to test whether ATX induction by TSA is limited to SW480 cells. TSA induced ATX expression in various cancer cell types, including breast, cervical, lung, ovarian, and prostate cancer cells Figure . FurtherTSA inhibits the Class I and II, but not Class III HDACs. More specifically, TSA strongly inhibits HDAC1, HDAC2, HDAC3, HDAC4, HDAC6 and HDAC7 . To idenTo expand our studies on the role of HDACs in ATX expression, we examined expression levels of HDAC3, HDAC7 and ATX in 13 cancer cell lines. The levels of HDAC3 expression were almost similar in all of cell lines tested, while the levels of HDAC7 expression had an inverse correlation with those of ATX expression. In particular, in 10 cancer cells with relatively high HDAC7 expression, the endogenous ATX expression is low or undetectable , indicating that LPA2 is involved in the protective effect of ATX-LPA signaling.The biological function of ATX is to convert LPC into LPA, a bioactive lysophospholipid that acts on specific G protein-coupled receptors (GPCRs) to perform various biological activities, including the anti-apoptosis activity. Quantitative PCR analyses showed that LPAs Figure . Down-reA Figure , while d1 was the dominant LPA receptor expressed , indicating that ATX-LPA axis protects MDA-MB-231 cell from the TSA-induced apoptosis through the LPA1-mediated signaling. Collectively, our data suggest that the cell-specific LPA receptor(s) is involved in the LPC-ATX-LPA signaling axis, which plays a role in reducing the efficacy of apoptosis induction by TSA and potentially other HDACi.Similar to SW480 cells, MDA-MB-231 cells were sensitive to TSA and were protected by LPA and LPC from the TSA-induced apoptosis (data not shown). In MDA-MB-231 cells, LPAd Figure . The pro1 Figure , but not50 in nanomolar range . 18:1 lysophosphatidylcholine (LPC) and 18:1 lysophosphatidic acid (LPA) were from Avanti Polar Lipid Inc. . The HDAC7 expression plasmid was a kind gift from Dr. Hung-Ying Kao [5-AZA-2'-deoxycytidine, trichostatin A (TSA), sodium butyrate (NaB), and Valproic acid (VPA) were purchased from Sigma-Aldrich . FS-3 (lysoPLD/Autotaxin substrate), 1-Bromo-3(S)-hydroxy-4- butylphosphonate (BrP-LPA) and 4-(Tetradecanoylamino) benzyl phosphonic acid disodium salt  were obtained from Echelon Biosciences . LPAYing Kao , and theYing Kao .The ATX primary antibody was generated as described previously . The pri2. For experiments to detect the effects of LPA and LPC on cell apoptosis, the cancer cells were cultured in a conditional serum-free medium with 250 μg/ml fatty-acid free BSA as described previously [1-GAAAUGAGCGCCACCUUUA; LPA2-GGUCAAUGCUGCUGUGUAC; LPA3-CAGCAGGAGUUACCUUGUU; HDAC1-GCUUCAAUCUAACUAUCAA; HDAC2-CAGUGAUGAGUAUAUCAAA; HDAC3-GCCGGUUAUCAACCAGGUA; HDAC4-CGUCAACAUGGCUUUCACC; HDAC6-GCUCGGCCAAGCAAUGGAA; HDAC7-UCACUGACCUCGCCUUCAA; non-specific-UUCUCCGAACGUGUCACGU. The siRNAs were transfected into cells with lipofectamine 2000 (Invitrogen), according to the protocol supplied by manufacturer. In each siRNA transfection experiment, the non-specific siRNA was used as control.The SW480, MDA-MB-231, MCF-7, MDA-MB-468, Hela, A549, MDA-MB-435 and HT-29 cells were cultured in Dulbecco's modified Eagle's medium (Hyclone). A2780, HEY, OCC1, PC-3, Du145, and colo320 cells were maintained in RPMI-1640 medium (Hyclone). Mediums were supplemented with 10% fetal bovine serum (Hyclone), 2 mM L-glutamine (Gibco), 100 μg/ml streptomycin (Gibco) and 100 U/ml penicillin (Gibco). All cells were cultured at 37°C in a humidified atmosphere containing 5% CO1 primers:5'-CGGCGGGTAGTGGTGGTC-3' and 5'-GTCGCGGTAGGAGTAAATGATG-3'; LPA2 primers: 5'-GTCGAGCCTGCTTG TCTTCC-3' and 5'-CCAGGAGCAGTACCACCTG-3'; LPA3 primers: 5'-GACGGTGA TGACTGTCTTAGGGG-3' and 5'-GAGGACTGTGGAGGGGATGC-3'. The PCR conditions were: 20 s at 95°C, 20 s for annealing, 25 s at 72°C for reasonable cycles, with a final extension for 5 min at 72°C. Each RT-PCR experiment was repeated at least three times with 3 parallel samples. The real-time RT-PCR was performed using the iQ SYBR Green Supermix (Bio-Rad) with the iCycler iQ real-time RT-PCR detection system (Bio-Rad). Relative expression of each target gene was estimated by normalization with the expression of GAPDH.Total RNA was extracted from cancer cells with Trizol (Invitrogen), and then digested with DNase I (Ambion) for 15 min at 37°C in order to remove DNA contamination. RNA (2 μg) from cancer cells were reverse-transcribed using anchored oligo dT (15) primers and the Reverse Transcription System (Promega). The cDNAs encoding indicated genes were amplified with specific primers. ATX primers: 5'-TATGCTTCGGAAAGAAATGGAG-3' and 5'-ATGTTCAATGTCACGCACCCT-3'; GAPDH primers: 5'-TTAGC ACCCCTGTCCAAGG-3' and 5'-CCTACTCCTTGGAGGCCATG-3'; HDAC1 primers: 5'-GAACTGGGGACCTACGGG-3' and 5'-GCTCTTGACAAATTCCACACAC-3'; HDAC2 primers:5'-AGTTGCCCTTGATTGTGAGA-3' and 5'-CCACTGTTGTCCTTGGATTTAT-3'; HDAC3 primers:5'-TGATGACCAGAGTTACAAGCAC-3' and 5'-GGGCAACATTTC GGACAG-3'; HDAC4 primers:5'-GTGCTGGTGTCATCAGGCTT-3' and 5'-AAATGGCG GTCAGGTCGT-3'; HDAC6 primers:5'-TGCTGTGACACCACTGCCC-3' and 5'-TTCT GGTGGGCGATGTTCTT-3'; HDAC7 primers 5'-GGATTTGATGCTGCTGAGGG-3' and 5'-CCACAGAGAGGGACGCCAG-3'; LPAThe conditional serum-free medium from cancer cells with or without exposure to TSA was concentrated (30-fold) using Amicon Ultra 30,000 (Millipore). The lysoPLD activity in the concentrated conditional medium was analyzed using fluorogenic substrate FS-3 as described previously . BrieflyLipids were extracted from concentrated (30-fold) conditional medium of SW480 cells with or without TSA treatment, and analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described previously .Cells were lysed in RIPA buffer for 30 min. After centrifugation, the supernatants were quantified by bicinchoninic acid assay . For experiments detecting the secreted ATX protein, the culture medium was concentrated (by approximately 30-fold) using Amicon Ultra 30,000 (Millipore). Protein quantification was conducted and equal amount protein was loaded for each sample. Protein samples were subjected to SDS-PAGE and analyzed as described previously . Each We3) after washing. NaCl was added into eluted samples  to reverse histone-DNA cross-links and the samples were heated for 4 hrs at 65°C. The purified DNA was used for PCR reactions. Primers for PCR amplification of the ATX promoter DNA were 5'-ATGATAGCTTAAGCCTCTTAGG-3' and 5'-TGCAGCGTGTTCTCTTTGCCTT-3'. PCR was carried out for 35 cycles , and PCR products were resolved on 2% agarose gel.ChIP assay was performed with a commercial Kit (Millipore 17-371). Briefly, formaldehyde was added to the medium to a final concentration of 1% and incubated for 10 min at room temperature, followed by addition of glycine (0.125 M) and incubation for another 5 min. The fixed cells were scraped into conical tubes, pelleted, and lysed in a SDS lysis buffer containing protease inhibitor (Roche). DNA was sheared to fragments of 500-1000 bp by sonication. The chromatin was precleared with salmon sperm DNA/protein G-agarose slurry for 90 min at 4°C. The precleared supernatant was incubated with antibodies against acetylation histone H3 (K9), histone H3 or normal rabbit IgG overnight at 4°C. The immunocomplexes were eluted with elution buffer  p < 0.005, (**) p < 0.001.ATX: Autotaxin; TSA: trichostatin A; HDAC: Histone deacetylase; HDACi: HDAC inhibitor; LysoPLD: lysophospholipase D; BrP: LPA-butylphosphonateThe authors declare that they have no competing interests.Conceived and designed the experiments: JZ. Performed the experiments: SL and BW; Data analyzed: SL, YX and JZ. Paper writing: SL, YX and JZ. All authors have read and approved the final manuscript.figure S1 - HDAC inhibitors, NaB and VPA, upregulate ATX expression in different cancer cells. Hela, MDA-MB-231 and Du145 cells were treated with NaB (1 mM) or VPA (1 mM) for 24 hrs, and then the ATX mRNA expression levels were evaluated by RT-PCR.Click here for filefigure S2 - Knockdown of individual HDAC alone could not up-regulate ATX expression. SW480 cells were transfected with the indicated HDAC siRNA with non-specific siRNA (siNC) as control. Total RNA was extracted at 48 hrs post transfection, and then the mRNA expression levels of HDACs and ATX in SW480 cells were detected by RT-PCR.Click here for filefigure S3 - BrP-LPA and S32826 inhibited ATX lysoPLD activity. SW480 cells were treated with or without TSA (100 nM) for 24 hrs. After treatment, the conditional culture medium was collected and concentrated about 30-fold. Different doses of BrP-LPA (A) or S32826 (B) were added to the concentrated conditional medium as indicated. After incubation for 1 hr, LysoPLD activity in the conditional medium was determined with FS-3 as substrate as described in Methods.Click here for filefigure S4 - ATX inhibitor S32826 enhanced the TSA-induced cell apoptosis. The SW480 (A), MDA-MB-231 (B), colo320 (C) and MDA-MB-435 (D) cells were pretreated with or without ATX inhibitor S32826 (1 μM) for 1 hr, and then treated with TSA (250 nM) in the presence of LPC (100 μM) or LPA (5 μM) as indicated. The cell apoptosis was measured after TSA treatment for 24 hrs. The p values derived from Student's t test are (*) p < 0.005, (**) p < 0.001.Click here for filefigure S5 - Histone acetylation in ATX promoter region is insufficient to up-regulate ATX expression. A, SW480 and HT-29 cells were treated with or without TSA (100 nM) for 24 hrs, and then subjected to ChIP assays as described in Methods to detect the acetylated histone H3 in the ATX promoter region. ATX expression levels were detected by RT-PCR. B, SW480 cells were transfected with HDAC3 and/or HDAC7 siRNA(s) as indicated. The ChIP assays were performed 48 hrs post transfection to detect the acetylated histone H3 in ATX promoter region. The acetylation level of histone H3 (Lys9) in SW480 cell lysates was detected by Western blot analyses and normalized by the total histone H3.Click here for filefigure S6 - Inhibition of ATX-LPA signaling enhanced TSA-induced apoptosis in serum-containing medium. The MDA-MB-231 cells were treated with TSA (1 μM) for 48 hrs in the conditional serum-free medium or serum (10%)-containing medium to detect the effect of serum on TSA-induced apoptosis. Furthermore, in serum (10%)-containing medium, MDA-MB-231 cells were pretreated with ATX inhibitor S32826 (1 μM) or LPA1/3 inhibitor Ki16425 (1 μM) for 1 hr, and then treated with TSA (1 μM) for 48 hrs. The cell apoptosis was measured after TSA treatment. The p value derived from Student's t test is (**) p < 0.001.Click here for file


A 67-year-old woman with chronic bilateral intraocular inflammation associated with vitreous cells and subretinal pigment epithelium (RPE) infiltrates (A–B) underwent a chorioretinal biopsy in the left eye after three negative vitreous biopsies. Sub-RPE deposits on spectral domain optical coherence tomography (C) corresponded to collections of diffuse large B-cell lymphoma cells on histopathogy (D–E) and immunohistochemistry (F–H).


Homeobox genes encode transcription factors impacting key developmental processes including embryogenesis, organogenesis, and cell differentiation. Reflecting their tight transcriptional control, homeobox genes are often embedded in large non-coding, cis-regulatory regions, containing tissue specific elements. In T-cell acute lymphoblastic leukemia (T-ALL) homeobox genes are frequently deregulated by chromosomal aberrations, notably translocations adding T-cell specific activatory elements. NKX3-1 is a prostate specific homeobox gene activated in T-ALL patients expressing oncogenic TAL1 or displaying immature T-cell characteristics. After investigating regulation of NKX3-1 in primary cells and cell lines, we report its ectopic expression in T-ALL cells independent of chromosomal rearrangements. Using siRNAs and expression profiling, we exploited NKX3-1 positive T-ALL cell lines as tools to investigate aberrant activatory mechanisms. Our data confirmed NKX3-1 activation by TAL1/GATA3/LMO and identified LYL1 as an alternative activator in immature T-ALL cells devoid of GATA3. Moreover, we showed that NKX3-1 is directly activated by early T-cell homeodomain factor MSX2. These activators were regulated by MLL and/or by IL7-, BMP4- and IGF2-signalling. Finally, we demonstrated homeobox gene SIX6 as a direct leukemic target of NKX3-1 in T-ALL. In conclusion, we identified three major mechanisms of NKX3-1 regulation in T-ALL cell lines which are represented by activators TAL1, LYL1 and MSX2, corresponding to particular T-ALL subtypes described in patients. These results may contribute to the understanding of leukemic transcriptional networks underlying disturbed T-cell differentiation in T-ALL. T-cells derive from early progenitor cells originating from hematopoietic stem cells in the bone marrow. After emigrating, T-cell progenitors complete their development in the thymus as thymocytes. Several pathways are crucial for developmental processes of thymocytes: these include BMP4, IGF2, IL2, IL4, IL7, IL15, NOTCH, TGFb and WNT signalling pathways Homeodomain proteins regulate fundamental differentiation processes in embryogenesis and the adult. Members of the HOX-family  and of the NKL-family  are active in the development of T-cells In T-cell acute lymphoblastic leukemia (T-ALL) thymocyte differentiation is disturbed, resulting in leukemic cells developmentally arrested at particular stages. These cells express certain oncogenes which subsequently serve as indicators for classification of T-ALL subtypes Many oncogenes identified in T-ALL encode factors regulating early stage specific thymocyte development , or ectopically activated factors  NKX3-1 is a member of the NKL-family of homeobox genes and is physiologically expressed in developing and mature prostate Here, we analyzed the deregulated expression of homeobox gene NKX3-1 in T-ALL cell lines. The aim of the study was to identify upstream and downstream activities of leukemic NKX3-1. Our data indicate absence of chromosomal aberrations and of ectopic prostate-specific impacts and illustrate particular pathways and factors activating leukemic NKX3-1 transcription.Cell lines are held by the DSMZ  except PER-117 generously provided by Ursula Kees, Perth, Australia Fluorescent in-situ-hybridization (FISH) analyses were performed as described previously https://cabig.nci.nih.gov/community/caArray_GSKdata/). The datasets for PER-117 (100 K array) were generated at the Helmholtz Centre for Infection Research . Genotyping and unpaired copy number analysis were performed using 30 reference data sets as normal control downloaded from Affymetrix. Copy number analyses were performed using the Affymetrix Genotyping Console GTC Software version 4.0  and visualized by the Affymetrix GTC-Browser program.For genome-wide copy number analysis of cell lines JURKAT and PER-117 we used the Affymetrix platform. The datasets for JURKAT (500 K array) were obtained from the National Cancer Institute , GSK Cancer Cell Line Genomic Profiling Data . Peripheral blood mononuclear cells (PBMC) were isolated from healthy volunteers (coauthors of this paper) using Lymphoprep . Primary CD19-positive B-cells were prepared from PBMC using corresponding magnetic beads from Miltenyi Biotec . Subsequently, we determined the purity of CD19-positive cells at 85% by flow cytometry. Primary CD3-positive T-cells were provided by Prof. Dr. Michaela Scherr  isolated from healthy donors, using magnetic beads from Miltenyi Biotec. Total human RNA isolated from bone marrow, lymph nodes, thymus, prostate, and retina was obtained from Clontech . Murine tissues, including bone marrow, popliteal lymph node, spleen, thymus, prostate, and eye were prepared from wild type C57BL/6 mice . 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  or oligonucleotides obtained from MWG Eurofins  as listed in Western blot analysis was performed by the semi-dry method. Proteins obtained from cell lysates were transferred onto nitrocellulose membranes  which were blocked with 5% dry milk powder dissolved in phosphate-buffered-saline buffer (PBS). The following antibodies were obtained from Invitrogen: anti-NKX3.1 (clone 3–9), from Santa Cruz Biotechnology: anti-ERK1 (K-23), GATA2 (H-116), GATA3 (HG3-31), and from Abnova : anti-LYL1 (B01P). The secondary antibodies have been connected with peroxidase and were detected by Western Lightning-ECL .ChIP analysis was performed with the ChIP Assay Kit  as reported previously HindIII/BamHI and EcoRI sites, respectively, of the expression vector pcDNA3 downstream of the CMV enhancer For creation of the reporter gene constructs we combined a reporter gene with genomic fragments, containing binding sites for MSX2 or NKX3-1. We cloned genomic PCR products (oligonucleotides are listed in www.ncbi.nlm.nih.gov/gds). 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). For statistical analyses the primary expression data (CEL-files) were processed using the R-based Bioconductor package before comparing NKX3-1 positive and negative T-ALL cell lines using the LIMMA package For quantification of gene expression via profiling, we used 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 . However, no chromosomal rearrangements were detected, indicating a wild type configuration throughout. We then analyzed copy number variations in JURKAT and PER-117 by genomic profiling. Again in both cell lines no changes in genomic copy number at the NKX3-1 locus at 8p21 were detected . Nevertheless, the NKX3-1 positive cell line RPMI-8402 expressed the highest levels of FOXA1 as analyzed by RQ-PCR . SiRNA mediated knockdown of FOXA1, ETS1 and SOX4 in RPMI-8402 and JURKAT cells inhibited the targeted TFs, while NKX3-1 was spared . Accordingly, JURKAT cells were treated with BMP4, TGFbeta, FGF9, NOTCH-inhibitor DAPT, WNT5B, and ATRA. However, none of these succeeded in inducing any increase in NKX3-1 expression as analyzed by RQ-PCR. Indeed, BMP4 and ATRA reduced expression of NKX3-1 . Expression of TAL1 was detected in 11 cell lines, 6 of which also expressed NKX3-1 , activating LMO1 Fig. 3A.Using siRNA mediated knockdown of particular TFs and subsequent quantification of NKX3-1 expression by RQ-PCR we were able to measure their likely impact on transcriptional activity. Knockdown of LMO1 in JURKAT cells and of LMO2 in MOLT-14 cells resulted in reduced expression of LMO1/2 and NKX3-1, confirming the activatory impact of LMO proteins in TAL1 positive T-ALL cells Fig. 3D.Next we analyzed the role of GATA-factors in NKX3-1 regulation. SiRNA mediated knockdown and overexpression of GATA3 in JURKAT demonstrated an activatory role Fig. 3F.Accordingly, siRNA mediated knockdown of MLL in JURKAT cells boosted expression of TAL1, GATA3, LMO1 and subsequently that of NKX3-1 Fig. 3I.To characterize additional upstream activators and to identify downstream targets of NKX3-1 we analyzed expression profiling data of T-ALL cell lines. Comparison of five NKX3-1 positive cell lines with four NKX3-1 negative ones using the R-based LIMMA package revealed 209 probe sets of differentially expressed genes (p<0.003). Consistently, the upregulated genes included NKX3-1 and TAL1 Fig. 4AFig. S4A). In comparison to the control, stimulation of CD3 resulted in enhanced NKX3-1 expression. Moreover, coactivation of GATA3, LMO1 and TAL1 indicates NKX3-1 regulation by TCR/CD3 via this TF complex (TRBC1 (T-cell receptor beta constant 1), IFT57 (intraflagellar transport 57) and NCS1  were upregulated and UBASH2/STS1 (ubiquitin associated and SH3 domain containing 2) was downregulated in NKX3-1 positive cell lines and involved in TCR signalling  complex Fig. 4B.Fig. S4B). Their potential impact on NKX3-1 expression was analyzed by treatment of PER-117 and JURKAT cells with IL2, IL4, IL7, IL13, IL15 and IGF2. In PER-117 IL13 and IGF2 treatments resulted in increased NKX3-1 expression while the other stimulations showed no effect . Stimulation of JURKAT cells with IGF2 resulted in no significant change of GATA3, LMO1 or TAL1 expression (IL15 (interleukin 15), CTBP2 , TFCP2 (transcription factor CP2) and IGF2BP1 (insulin-like growth factor 2 mRNA binding protein 1) are upregulated and RUNX3 (runt-related transcription factor 3) downregulated in NKX3-1 positive cell lines. These genes are involved in IL/JAK/STAT- and IGF2-pathways Table 3 MSX2 has been described as a target gene of IGF-signalling in odontoblasts and may play a role in T-cell development , www.genome.cse.ucsc.edu), the SIX6 gene contains a potential binding site for NKX3-1 in exon 2. Subsequent reporter gene assay using the corresponding genomic fragment confirmed direct activation of SIX6 by NKX3-1  using flanking and straddling probes of the NKX3-1 locus indicate wild type configurations.(TIF)Click here for additional data file.Figure S2Expression analysis of TFs and signalling components. (A) Data of expression analyses obtained by profiling for genes encoding TFs NKX3-1, FOXA1, SOX4 and ETS1 in T-ALL cell lines were transformed into a heat map (above). Red indicates high expression levels, black medium and green low levels. Expression analyses of FOXA1 by RQ-PCR in T-ALL cell lines (left) and in three prostate cell lines in comparison to RPMI-8402 (right) show elevated levels in RPMI-8402, which are, however, much lower when compared to prostate cells. (B) Heat map of expression profiling data from T-ALL cell lines demonstrates expression intensities of signalling-pathway components.(TIF)Click here for additional data file.Figure S3LMO4 expression. Quantification of gene expression for LMO4 in T-ALL cell lines by RQ-PCR. NKX3-1 expressing cell lines are indicated in red letters.(TIF)Click here for additional data file.Figure S4Analysis of signalling components. (A) Expression analyses of TCR and CD3 genes. The heat map (above) displays expression profiling data of T-ALL cell lines. Red indicates high expression levels, black medium and green low levels. Flow cytometry data (below) demonstrates significant CD3 protein expression in JURKAT (92%) but absent from PER-117. (B) The heat map shows expression profiling data of T-ALL cell lines for several IL/STAT- and IGF/IGFR-genes. (C) PER-117 cells were treated for siRNA mediated knockdown of STAT5A and STAT3. Collectively, the data indicate STAT5-mediated inhibition of GATA2, GATA3, LYL1 and NKX3-1.(TIF)Click here for additional data file.Table S1Potential NKX3-1 regulator- and target-genes.(DOC)Click here for additional data file.


INK4A, which sensitizes ALL cells to mitochondria-induced cell death, represses FOXO3 protein levels and reduces the dependency of these leukemia cells on PI3K-PKB signaling. This suggests that if p16INK4A is deleted during leukemia development, FOXO3 levels elevate and FOXO3 has to be inactivated by deregulation of the PI3K-PKB pathway to prevent FOXO3-induced cell death.Forkhead transcription factors (FOXO) are downstream targets of the phosphoinositol-3-kinase (PI3K) protein kinase B (PKB) signaling cascade and play a pivotal role in cell differentiation, cell cycle and apoptosis. We found that cells from prednisone-resistant T-acute lymphoblastic leukemia (T-ALL) patients showed cytoplasmic localization of FOXO3 in comparison to prednisone-sensitive patients suggesting its inactivation. To determine the impact of FOXO3, T-ALL cells were infected with a 4OH-tamoxifen-regulated, phosphorylation-independent FOXO3(A3)ERtm allele. After FOXO3-activation these cells undergo caspase-dependent apoptosis. FOXO3 induces the death ligand TRAIL and the BH3-only protein Noxa implicating extrinsic as well as intrinsic death signaling. Whereas dnFADD partially inhibited cell death, CrmA and dnBID efficiently rescued ALL cells after FOXO3 activation, suggesting a caspase-8 amplifying feedback loop downstream of FADD. Knockdown of TRAIL and Noxa reduced FOXO3-induced apoptosis, implicating that mitochondrial destabilization amplifies TRAIL-signaling. The-reconstitution of the cell cycle inhibitor p16 Forkhead transcription factors (FOXO) are downstream targets of the phosphoinositol-3-kinase (PI3K) protein kinase B-(PKB)-signaling pathway . The FOXOne explanation for PKB hyperactivation might be that T-ALL cells frequently carry a heterozygous deletion of the PTEN gene locus , which lFOXO3 was shown to play a crucial role in controlling cell cycle arrest, apoptosis and self-renewal of haematopoietic progenitor cells , 12 and c is released from mitochondria and triggers the formation of the apoptosome, which leads to subsequent activation of CASP9/caspase-9. Two models have been discussed how BH3-only proteins induce cell death. The first model describes activators of BAX and BAK, like Bid and Bim, and sensitizers like Noxa which bind to anti-apoptotic proteins and thereby release activator BH3-only proteins as well as BAX and BAK [Apoptosis is initiated either by binding of so called death ligands, such as FASL or TRAIL to their cognate receptors resulting in formation of the death-inducing signaling complex (DISC) and activation of CASP8/caspase-8, or via the intrinsic death pathway. The intrinsic death pathway is controlled at the level of mitochondria by members of the BCL2 family. This protein family is divided into anti-apoptotic members, such as BCL2, BclxL or MCL1, the pro-apoptotic BH3-only proteins  and the pro-apoptotic multi-domain proteins BAX, BAK and Bok. The balance of pro- and anti-apoptotic proteins (BCL2 rheostat) regulates mitochondrial membrane potential. If damaged, Cytochrome  and BAK . The secThe BH3-only protein BID connects the extrinsic and intrinsic signaling, because it is cleaved by active caspase-8 and then inserts into the outer mitochondrial membrane where it antagonizes the function of the pro-survival BCL2 proteins. In some cell types  extrinsic death signaling always involves amplification of the death signal via mitochondria, since overexpression of either BCL2 or BclxL prevents TRAIL-induced apoptosis in CEM cells . This isINK4A in T-ALL represses endogenous FOXO3, suggesting that these two tumor suppressor proteins cooperate to prevent childhood leukemia.In this study we investigated whether therapy resistance in childhood T-ALL cells correlates with inactivation of FOXO3. We uncovered, that FOXO3 activates apoptosis by induction of TRAIL and Noxa and found that the expression of the frequently mutated tumor suppressor p16Deregulation of the PI3K/PKB/FOXO3 pathway was shown to be involved in cancer development and contributes to therapy resistance of different malignancies. Bone marrow cells from pediatric T-ALL patient samples were divided into good responders to initial prednisone therapy (PGR) and prednisone poor responders (PPR) and were analyzed by immunofluorescence for FOXO3 expression and subcellular localization. As shown in Fig As FOXO3 activation leads to apoptosis induction in haematopoietic cells , 12, we Kip1 and/or p21Cip1 [Depending on the cell type FOXO3 induces cell cycle arrest by elevating the expression of the cell cycle inhibitors p27 p21Cip1 , 24 or t p21Cip1  or membe p21Cip1 , 18, 25. p21Cip1 . The staTo analyze how the inhibition of key-steps in extrinsic and intrinsic death signaling affects cell survival of T-ALL during FOXO3 activation, we measured the number of apoptotic cells by FACS-analyse of PI-stained nuclei. FOXO3 activation by 4OHT induced 47% apoptosis after 24 hours – the expression of CrmA (CEM/FOXO3-CrmA) and dnBID (CEM/FOXO3-dnBID) reduced the apoptosis rate to 15.2% and 14.5%, respectively. Overexpression of BclxL prevented apoptotic cell death almost completely (11.5%), whereas transgenic BCL2 lowered cell death to 25.3% and dnFADD reduced apoptosis by 10% Fig . Also inWe next investigated potential FOXO3 targets that may be crucial for cell death initiation. We activated FOXO3 in CEM/FOXO3, Molt3/FOXO3 and Molt4/FOXO3 cells for 6 hours with 4OHT and analyzed the expression of several death inducers of the extrinsic and intrinsic cell death signaling pathways. Among them we identified TRAIL and the BH3-only proteins Bim and Noxa as FOXO3-regulated targets. TRAIL mRNA was induced up to 50fold after 6 hours, whereas e.g. FASL was not regulated Fig , suggestINK4A sensitizes leukemia cells to mitochondrial cell death by changing the balance of pro- and anti-apoptotic BCL2 proteins at the mitochondria [INK4A and FOXO3 might be connected in ALL cells. As shown in Fig INK4A induces cell cycle arrest in the G1 phase and represses FOXO3 steady state expression levels within 24 hours ER fusion protein and validated the expression by immunoblot ER allele is activated by 4OHT in p16INK4A-expressing cells, p16INK4A further increases cell death . All reagents were purchased from Sigma-Aldrich  unless indicated otherwise. For each experiment, mid-log phase cultures were seeded in fresh medium.Jurkat, Molt3, Molt4, CEM/C7H2 (CEM), a subclone of the CCRF/CEM-C7 cell line and all transgenic variants were cultured in RPMI 1640  containing 5% fetal calf serum , 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM L-glutamine  at 5% COd before . Phoenix4Cl (Sigma-Aldrich) 50 mM for 15 min to reduce background.Primary T-ALL cell culture were placed on the slide by cytospin and then fixed with formaldehyde (4%) for 15 min. Slides were treated with NHCells were permeabilized with 0.1% Triton X-100/phosphate buffered saline for 3 min and incubated first with blocking buffer  and then with monoclonal FOXO3 antibody  diluted in blocking buffer overnight at 4 °C. Cells were then incubated for 1 h with specific secondary antibody conjugated to Alexa Fluor 488 , and for 10 min with 4.6-diamidino-2-phenylindole (DAPI) 1:10000; Sigma-Aldrich, Vienna, Austria). Images were acquired by the videoconfocal system ViCo microscope Nikon Eclipse 80i .T-ALL patient samples were obtained after informed consent following the tenets of the Declaration of Helsinki. Diagnosis was made according to standard cytomorphology, cytochemistry and immunophenotypic criteria. All analyzed T-ALL samples were obtained at the time of diagnosis before treatment, after Ficoll–Hypaque  separation of mononuclear cells. Mononuclear cells were frozen as viable cells in FCS and 10% DMSO and stored in liquid nitrogen. The percentage of CD7 cells ranged from 85 to 96%.5 Phoenix™ packaging cells were transfected with 2 µg of retroviral vector and 1 µg of a plasmid coding for VSV-G protein using Lipofectamine2000 . Following vectors have been described previously: pLIB-FOXO3(A3)ERtm-iresNeo, pLIB-BCL2-iresPuro, pLIB-BclxL-iresPuro, pLIB-CrmA-iresPuro, pLIB-dnBID-iresPuro, pLIB-dnFADD-iresPuro, pQ-tetH1-shBim-SV40-Puro, pQ-tetH1-shNoxa-SV40Puro, pLIB-ECFP-FOXO3wt-iresPuro, pLIB-MCS2-iresPuro, pLIB-MCS2-iresNeo and pQ-tetH1-SV40Puro [BamH1-Mun1 sites of the plasmid pQ-tetH1-SV40-Puro. Plasmids were transfected into Phoenix packaging cells to generate supernatants. After 48h, the retrovirus-containing supernatants were filtered  and used to infect CEM cells. The resulting cell lines are: CEM/FOXO3-Ctr, CEM/FOXO3-BCL2, CEM/FOXO3-BclxL, CEM/FOXO3-CrmA, CEM/FOXO3-dnFADD, CEM/FOXO3-dnBID, CEM/FOXO3-shCtr, CEM/FOXO3-shBim, CEM/FOXO3-shNoxa, CEM/FOXO3-shTRAIL, CEM/p16-ECFP-FOXO3wt and CEM/p16-FOXO3(A3)ER.Approximately 6x10SV40Puro , 34, 35.5 cells were centrifuged and resuspended in PI solution containing 0.1% Triton X-100. Stained nuclei in the sub-G1 region were considered to represent apoptotic cells. Mitochondrial activity and membrane potential were assessed by the fluorescence dye MitoTracker Red/CMX-Ros . For measurement of AnnexinV positive cells 5x105 cells were harvested and stained with FITC-labeled AnnexinV  in Annexin Binding Buffer.Apoptosis was assessed by staining the cells with propidium-iodide (PI) or AnnexinV using a CytomicsFC-500 Beckman Coulter as previously described : 2x105 c6 cells were lysed on ice in lysis-buffer  with protease and phosphatase inhibitors. Protein concentration was measured using Bradford-Reagent . Equal amounts of total protein (50 μg/lane) were separated by SDS-PAGE and transferred to nitrocellulose membranes  by a semi-dry blotting device . Membrane blocking was performed with PBS blocking buffer containing 0.1% Tween20 and 5% nonfat dry milk, incubated with primary antibodies specific for human BclxL, caspase-3, BID, FOXO3, , BCL2, Bim, CrmA, caspase-8, FADD , Noxa , TRAIL , caspase-9  and α-Tubulin  then washed and incubated with anti-mouse, anti-rabbit or anti-rat horseradish-peroxidase-conjugated secondary antibodies . The immunoblots were developed by enhanced chemiluminescence  according to the manufacturer´s instructions and analyzed in an AutoChemi detection system .5x106 cells using TRIzol™ Reagent  according to the manufacturer´s instructions. Complementary DNA was synthesized from 1 µg of total RNA using the RevertAid™ First Strand H minus cDNA Synthesis Kit . The oligonucleotides to amplify mRNA fragments were Bim , Noxa , TRAIL , and GAPDH  and were synthesized by Microsynth AG . After normalization on GAPDH expression, regulation was calculated between treated and untreated cells. Control is set as 100% expression.To quantify Bim, Noxa, and TRAIL mRNA levels we designed real-time RT-PCR assays, using GAPDH as reference gene. Total RNA was isolated from 5x10Statistical significance of differences between controls and treated cells were calculated using unpaired t-test. All statistical analyses were performed using Graph Pad Prism 4.0 software.


Enterobacter cloacae was isolated from blood culture. The strain showed multiple resistances to clinically used antibiotics, including all β-lactams, fluoroquinolones, aminoglycosides, and sulfonamides. Sequence analysis showed the presence of 14 genes associated with resistance to antibiotics, including the metallo-β-lactamase VIM-1 gene, which was located in a class 1 integron.The VIM-1-producing multidrug-resistant strain  Enterobacter cloacae is an important nosocomial pathogen and is intrinsically resistant to ampicillin and narrow-spectrum cephalosporins. Resistances to extended-spectrum cephalosporins and aztreonam are usually related to a mutational depression of the chromosomal Ambler class C β-lactamase or to the production of plasmid-mediated extended-spectrum β-lactamases  have been reported recently in several strains of E. cloacae , obtaining a total of 165,540,445 bp and 338,715 reads. The EC_38VIM1 assembly resulted in 90 contigs, with an N50 contig size of 262,093 nucleotides and a total length of 5,155,870 bp. Contigs were annotated using the Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) through NCBI (http://www.ncbi.nlm.nih.gov/), providing a total of 4,999 genes, 4,864 coding DNA sequence (CDS) genes, 56 pseudogenes, 4 rRNAs , and 75 tRNAs. Additionally, genome annotation was performed automatically on the Rapid Annotations using Subsystems Technology (RAST) server (http://arpcard.mcmaster.ca) , aminoglycosides (streptomycin 3′-adenylyltransferase [aadA1] and aminoglycoside 6-adenylyltransferase [aacA4]), fluoroquinolones (plasmid-mediated quinolone resistance [qnrA1]), chloramphenicol (chloramphenicol acetyltransferase [catB2]), and sulfonamides (dihydrofolate reductase [dfrB1]). Sequencing showed that the blaVIM-1 gene was contained in a class 1 integron. The structure of the integron was intI (integrase gene)-blaVIM-1-aacA4-dfrB1-aadA1-catB2-quacEΔ1/sul1 (quaternary ammonium compound resistance gene/sulfonamide resistance gene). An analysis of the genome of EC_38VIM1 identified the sequences of three plasmids, which belonged to the IncH1, IncF, and IncI1 groups. Overall, the availability of this genome sequence facilitates further comparative genomic analyses among E. cloacae strains with different antimicrobial susceptibility patterns in order to shed light on the classical and new antibiotic resistance mechanisms in this pathogen.Genomic DNA was extracted from an overnight culture using the DNeasy blood and tissue kit (Qiagen). Whole-genome shotgun sequencing of E. cloacae EC_38VIM1 has been included in the GenBank whole-genome shotgun (WGS) database under the accession no. ATHX00000000.The draft genome sequence of


The ALVAC-HIV/AIDSVAX-B/E RV144 vaccine efficacy trial showed an estimated efficacy of 31%. The immune correlates analysis raised the hypothesis that the observed protection in RV144 may be partially due to Antibody-Dependent Cellular Cytotoxicity (ADCC)-mediating antibodies in the presence of low levels of Env IgA antibodies. In this study we analyzed the Ig VH family usage of vaccine-induced ADCC mAbs isolated from memory B cells of vaccinees.From a total of 321,945 memory B-cells of 6 vaccinees we obtained 23 mAbs that mediated ADCC using IgG+ memory B-cell cultures (n=9) and Env-specific flow cytometric single memory B-cell sorting (n=14). ADCC activity was measured using both E.CM243 gp120-coated and E.CM235-infected target cells in a flow-based assay.ADCC-mediating mAbs displayed a disproportionate usage of VH1 family genes , in particular the VH1-2 gene segment , as recently observed for CD4bs broadly neutralizing antibodies (HAAD bNAbs). In contrast, only 17.1% of 111 heavy chains isolated from cultures that did not mediate ADCC used the VH1 gene. VH1 ADCC-mediating mAbs showed a high degree of V(D)J amino acid similarity to both the VH (68-84%) and VL (70-87%) HAAD motifs. V(D)J rearrangements displayed modest levels of affinity maturation (0.5-5.1% for heavy chains and 0.4-4.3% for light chains). While none of the VH1 ADCC-mediating mAbs was capable of mediating HIV-1 neutralization, the strength of their ADCC activity correlated with the levels of heavy chain somatic mutations (p=0.02). We produced the reverted unmutated ancestor antibodies of two VH1 ADCC-mediating mAbs: one bound to B.MN Env and both reacted against autoantigens.ADCC-mediating antibodies induced by the ALVAC-HIV/AIDSVAX-B/E vaccine underwent limited affinity maturation, and preferentially used VH1 gene segments which share the HAAD motif with CD4bs bNAbs. These observations raise the hypothesis that HIV-1 Env preferentially selects VH1 family usage for distinct subsets of antibodies with different functions.