Drosophila melanogaster genetic tool box includes many stocks for generating genetically mosaic tissue in which a clone of cells, related by lineage, contain a common genetic alteration. These tools have made it possible to study the postembryonic function of essential genes and to better understand how individual cells interact within intact tissues. We have screened through 201 enhancer-trap flippase lines to identify lines that produce useful clone patterns in the adult ovary. We found that approximately 70% of the lines produced clones that were present in the adult ovary and that many ovarian cell types were represented among the different clone patterns produced by these lines. We have also identified and further characterized five particularly useful enhancer-trap flippase lines. These lines make it possible to generate clones specifically in germ cells, escort cells, prefollicle cells, or terminal filament cells. In addition, we have found that chickadee is specifically upregulated in the posterior escort cells, follicle stem cells, and prefollicle cells that comprise the follicle stem cell niche region. Collectively, these studies provide several new tools for genetic mosaic analysis in the Drosophila ovary.The  Drosophila ovary is a tractable model for studying diverse biological processes, including oogenesis, tissue morphogenesis, and stem cell self-renewal. A common strategy for studying gene function in the ovary is to generate homozygous mutant cells by flippase (Flp)/FRT mitotic recombination. Often, Flp is expressed under the control of a heat shock promoter, which allows for precise control over the timing of clone induction, but it is not possible to target recombination to specific cell types or tissues with this method. Thus, mutant clones are generated in multiple cell types, making it more difficult to assess the cell-autonomous role of genes of interest. Another widely used approach has been to use a UAS-Flp construct and Gal4 lines that are expressed in specific tissues to activate Flp expression  is specifically upregulated in the FSC niche region. Finally, we provide a summary of the information for all 201 lines, which can be used as a resource for selecting additional lines of interest.Here, we report the results of the screen and highlight five lines that generate clones within specific subpopulations of cells in the ovary. We have verified the clone patterns for each of these five lines and mapped the ET-flpx2 insertion site for four lines. Furthermore, one line led us to the discovery that All stocks and crosses were maintained on standard molasses food at room temperature or 25°. In addition to the 201 ET-flpx2 lines, the following stocks were used:yw, actinP > cd2 > gal4; UAS-gfp3, ry506yw, P{Act5C(FRT.polyA)lacZ.nls1}w; Act-FRT-stop-FRT-lacZnls, Ubi-FRT-stop-FRT-GFPnls; Act-FRT-stop-FRT-GAL4, UAS-his2A::RFP/SM5-TM6B (TIE-DYE)hsFlp; FRT 40a, Ubi-GFPw; FRT40aTo obtain flies for ovary dissections, crosses were set up between the ET-flpx2 lines and one of the reporter stocks listed above. Once the progeny began to eclose, all adults were discarded and newly eclosed progeny were maintained on molasses food with fresh wet yeast daily for 2 d or 7 d.Ovaries were dissected in 1× Graces Medium with forceps, fixed in 4% paraformaldehyde, rinsed in 1× PBS plus 0.2% Tween (PBST), incubated overnight at 4° with primary antibody, washed for 1 hr with PBST, incubated at room temperature with secondary for 2 hr, washed for 1 hr with PBST, and incubated with PBS plus 1 ng/µl DAPI. Ovaries were then mounted on glass slides in vectashield and viewed using a Zeiss Axioimager for scoring and imaged with a Zeiss Apotome to obtain optical sections. The following primary antibodies were used: mouse anti-GFP , rabbit anti-GFP , rabbit anti-vasa , mouse anti-LacZ , rabbit anti-LacZ , and mouse anti-Fas3 (1:50) and mouse anti-chickadee (1:100) from the Developmental Studies Hybridoma Bank. The following secondary antibodies were used: anti-rabbit and anti-mouse conjugated to Alexafluor 488, 546, or 555 .hsFlp construct, flies of the appropriate genotype were cultured at 25° for at least 2 d after they eclosed, heat shocked at 37° four times over 2 d for 1 hr per heat shock, and cultured for at least 5 d to allow transient follicle cell clones to exit the germarium. Flies were given fresh wet yeast daily throughout the culture periods.To generate FSC clones in flies with the P-element). Oligonucleotide sequences were as follows:SPLNK-GATC-TOP GATCCCACTAGTGTCGACACCAGTCTCTAATTTTTTTTTTCAAAAAAASPLNK-BOT CGAAGAGTAACCGTTGCTAGGAGAGACCGTGGCTGAATGAGACTGGTGTCGACACTAGTGG SPLNK#1 CGAAGAGTAACCGTTGCTAGGAGAGACCSPLNK#2 GTGGCTGAATGAGACTGGTGTCGAC3′SPLNK#1 CACTCAGACTCAATACGACAC3′SPLNK#2 GGATGTCTCTTGCCGAC3′SPLNK-SEQ CGGGACCACCTTATGSplinkerette PCR was performed according to previously established protocol . GeneralP-element insertion at X:19586247, which is likely to be the original insertion site of the ET-Flpx2 before mobilization. Moreover, genetic analysis indicated that these lines both contain ET-Flpx2 insertions on an autosome. Therefore, we crossed away the X chromosome and redid the splinkerette reactions on the resulting mW+w flies. However, after multiple attempts using splinkerette PCR, we were still unable to obtain reliable information about the autosomal insertion sites in these two lines.PCR products were separated on a 1% agarose gel stained with ethidium bromide. Single bands were excised from the gel, and the DNA was purified and sequenced. Initial splinkerette PCR reactions for lines 168 and 688A both indicated a www.thebestgene.com), where inverse PCR was attempted according to standard procedures (http://www.fruitfly.org/about/methods/inverse.pcr.html). Multiple attempts to map the insertion in line 688A were unsuccessful, but two independent iPCR experiments mapped line 168 to an insertion site in the krasavietz gene. The genomic sequence returned by one of these iPCR experiments is provided below.Genomic DNA from lines 168 and 688A was sent to Best Gene, Inc. . We observed a similar clone frequency with both reporters , chic was upregulated in posterior escort cells, FSCs, and early prefollicle cells near the region 2a/2b border  and 68.2% (n = 58/85) of ovarioles at 2 dpe and 7 dpe, respectively. Specifically, we noticed that many of these clones were within the FSC niche region, which includes escort cells in region 2a, FSCs, and prefollicle cells in region 2b . We founchic , which hogenesis . We staifor chic . Becauseb border . This inDrosophila ovary. Previous screens identified LacZ and Gal4 enhancer trap lines with expression in the ovary (Our screen has identified a new set of lines for the study of oogenesis and stem cell biology in the he ovary , and sevts/Gal4/UAS-Flp or hsFlp. However, the ET-Flp2x lines still allow for spatially restricted clone induction while freeing the Gal4/UAS system for other uses. In addition, if the goal is to induce transgene expression in a subset of cells, then generating clones with an ET-Flp2x line may be a useful alternative to using a Gal4 enhancer trap in some cases because expression can be driven throughout the clone by a strong, uniform driver such as tub-Gal4 or ubi-Gal4 even if the ET-Flp2x line that provides the desired spatial specificity has relatively weak or variable expression levels.Clones induced by FRT recombination can also be used to identify cell types, such as stem cells, as well as to manipulate gene expression. Because the ET-Flp2x lines combine the features of an enhancer trap with Flp expression, they can be used much like Gal4/UAS-Flp combinations have been used previously. However, compared to existing tools, the ET-Flp2x lines have a distinct set of drawbacks and advantages. For example, the timing of Flp expression in ET-Flp2x lines cannot be controlled as it can be by temperature shifting flies with Gal80vs. region 2a escort cells. Likewise, line 820A will be useful for addressing questions about the specification of the polar/stalk lineage (Another advantage of ET-Flpx2 lines is the ability to label a group of cells in the adult tissue that shares a common progenitor. This is useful for studying the developmental relationships between cell types and for labeling subsets of cells within a uniform population. For example, line 688A may be useful for investigating whether there is functional diversity in the escort cell population. As a population, escort cells perform diverse functions in germ cell development and FSC self-renewal  but, in  lineage .chic is specifically upregulated in the FSC niche region. This establishes chic as a useful marker and suggests that it may be important for the function of the FSC niche. Chic is the Drosophila homolog of profilin (Finally, as with other types of enhancer traps, the ET-Flpx2 lines can be used to identify genes with interesting expression patterns. For example, our studies of line 398A led to the discovery that profilin . Escort profilin . It willDrosophila (in vivo context.The lines we have described here complement existing tools for genetic mosaic analysis in osophila . Althoug


Mechanisms underlying resistance development remain poorly understood and new therapeutic opportunities are urgently needed. Here, we report the generation and characterization of seven tamoxifen-resistant breast cancer cell lines from four parental strains.The estrogen receptor (ER) inhibitor tamoxifen reduces breast cancer mortality by 31 % and has served as the standard treatment for ER-positive breast cancers for decades. However, 50 % of advanced ER-positive cancers display Using high throughput drug sensitivity and resistance testing (DSRT) with 279 approved and investigational oncology drugs, exome-sequencing and network analysis, we for the first time, systematically determine the drug response profiles specific to tamoxifen resistance.We discovered emerging vulnerabilities towards specific drugs, such as ERK1/2-, proteasome- and BCL-family inhibitors as the cells became tamoxifen-resistant. Co-resistance to other drugs such as the survivin inhibitor YM155 and the chemotherapeutic agent paclitaxel also occurred.This study indicates that multiple molecular mechanisms dictate endocrine resistance, resulting in unexpected vulnerabilities to initially ineffective drugs, as well as in emerging co-resistances. Thus, combatting drug-resistant tumors will require patient-tailored strategies in order to identify new drug vulnerabilities, and to understand the associated co-resistance patterns.The online version of this article (doi:10.1186/s12885-016-2452-5) contains supplementary material, which is available to authorized users. Two-thirds of breast tumors express ER that drives proliferation of mammary epithelial cells and thereby contributes to the etiology and progression of the disease. Consequently, antagonists that directly block ER function or drugs that lower the amounts of the natural ligand of ER, estradiol, have been utilized in breast cancer treatment for decades . For ovey occurs . Some ofy occurs –5, altery occurs , 7, and y occurs . Consequy occurs , 10. Howy occurs . On the y occurs , 12. Bory occurs .Whilst shRNA- or cDNA-based functional screens , 15 and Here, we report the development and characterization of a panel of seven long-term tamoxifen-treated breast cancer cell lines from four parental strains. Using these resistant cell line models and their isogenic parental counterparts, we, for the first time, performed systematic high throughput drug sensitivity and resistance testing with 279 approved and investigational oncology drugs to reveal potential new drug vulnerabilities and to identify co-resistance patterns acquired with tamoxifen resistance. We further conducted exome-sequencing on each of the isogenic parental-resistant cell line pair to identify point mutations and copy number variations that may contribute to drug resistance. Through integrated network analyses, we uncovered cell- and clone-specific molecular and functional patterns of endocrine resistance, highlighting the underlying molecular diversity, and pointing to several distinct therapeutic opportunities to circumvent it. However, no systematic drug screens with hundreds of oncology compounds on acquired tamoxifen resistance have been conducted.2 and passaged when ca 80 % confluent. The approximate doubling times of the cells were as follows: parental MCF-7, T-47D, ZR-75-1 and BT-474: 1–3 days. Resistant MCF-7 Tam1, T-47D Tam1 and Tam2: 1–2 weeks, ZR-75-1 Tam1 and Tam2: > 1 week, BT-474 Tam1 and Tam2: 2 weeks. The cells were free of mycoplasma and verified for their authenticity .Human breast cancer cell lines MCF-7 , T-47D , ZR-75-1  and BT-474  were obtained from the American Type Culture Collection. The cells were grown in DMEM with L-Glutamine  or RPMI-1640 with L-Glutamine  supplemented with 10 % FCS  and 1 % penicillin/streptomycin (Gibco). Culture media for T-47D, MCF-7 and BT-474 additionally contained 0,1 % bovine insulin . The tamoxifen-resistant cell lines  were derived from the parental cell lines by continuous exposure to 4-OH-tamoxifen  for 8–12 months. Culture media was replaced every 2–3 days. All cells were incubated at 37 °C with 5 % CO−8 M in ethanol) was then added back to the cells for 4, 8 or 24 h and RNA isolated with Total RNA Purification kit . 4 μg of total RNA were reverse transcribed with the High-Capacity cDNA Reverse transcription kit  as instructed. Quantitative-PCR was then performed on the LightCycler 480 system  using the DyNAmo colour flash SYBRGreen PCR kit (Thermo Scientific) with equal amounts of cDNA. The optimal internal reference gene was determined out of a pool of 16 different housekeeping genes for each parental-resistant cell line pair . Primer sequences can be found in Additional file For viability measurements cells were seeded in 384-well culture plates with increasing tamoxifen concentrations 0–1,8 μM). After 120 h cell viability was evaluated by CellTiter-Glo Cell Viability Assay  with the PHERAstar plate reader . To measure the active DNA synthesis cell proliferation assays with Click-iT® EdU Alexa Fluor® 488 Flow Cytometry Assay Kit were performed according to manufacturer’s protocol (Life Technologies) with following minor modifications: Cells were plated on 10 cm plates and cultured with and without 1 μM tamoxifen until approximately 50 % confluent. Parental cells and their tamoxifen-resistant derivatives were then pulsed with 10 μM of EdU Alexa Fluor® 488 for 4 h (T-47D and MCF-7) or for 28 h (BT-474 and ZR-75-1). Cells were permeabilized with saponin-based permeabilization and wash reagent  or with 0,1 % TritonX-100-PBS (ZR-75-1) for 10 min. Additionally, DNA content staining was performed using FxCycle™ Far Red and the cell suspension treated with RibonucleaseA. Flow cytometry was carried out and results analyzed using Accuri C6 flow cytometer and associated software . To measure estrogen-responsivity of the parental cells and their tamoxifen-resistant derivatives, the cells were grown on 6-well plates in hormone-deprived culture medium for 72 h , supplemented with 2,5 % dextran–charcoal-treated (Sigma-Aldrich) FCS and other additives (see above). 17β-estradiol . Exome-capture was done on 3 μg DNA with the NimbleGen SeqCap EZ Human Exome v2.0 kit (Roche NimbleGen) and paired-end sequencing performed on Illumina HiSeq platform. Point mutations were detected as previously described , using th SnpEff  using thutations  were excutations . Plots outations –34 as foDSRT with the FIMM FO2Baq library containing 279 approved and investigational oncology drugs was done as previously described  with min50) values were extracted from the ChEMBL database (https://www.ebi.ac.uk/chembl), and integrated KIBA-score was calculated for each drug-target pair. A low KIBA-score indicates a high binding affinity of the drug with the target. Amongst the set of targets for a drug, if the target with highest binding affinity had KIBA-score of <0,1, the specific target threshold was considered to be 50-fold the lowest KIBA-value; otherwise KIBA-score <3 units was considered as the specific target threshold. Second, to capture the connection between genomic changes and drug-target genes of the most effective drugs in each resistant cell model compared to its parental cell line, network analysis and visualization was done on KIBA-scored specific targets genes of the sensitizing or desensitizing drugs using Ingenuity Pathway Analysis application . The IPA system is based on the Ingenuity Pathways Knowledge Base, which is a repository of curated biological interactions, and functional annotations between proteins, genes, complexes extracted from scientific articles. The network consists of thousands of nodes and the edges represent experimentally observed cause-effect relationships that are related to direct physical interactions or regulatory interactions events like expression, transcription, activation and molecular modifications. Network edges are also associated with a direction of the causal effect, i.e. either activating or inhibiting [www.qiagen.com) with the networks utilizing the IPA overlay tool.To visualize drug sensitivity and resistance networks in each resistant/parental cell line pair, drugs with DSS difference >5 (sensitivity) or < −5 (co-resistance) were selected. For each drug, specific target molecules were defined using the KIBA (Kinase Inhibitor BioActivity) -score  as follohibiting . Sensitihibiting . The IPATo study the development of tamoxifen resistance across distinct molecular backgrounds, we established seven long-term tamoxifen-treated cell lines originating from four parental cell types; MCF-7, T-47D, ZR-75-1 and BT-474. We exposed these ER-positive, initially tamoxifen-responsive cells to continuous 1 μM 4-OH-tamoxifen treatment (hereafter tamoxifen) for 8 to 12 months Fig. A. For fuTo uncover drug response profiles associated with long-term tamoxifen treatment mimicking the prolonged exposure to the drug used in the clinic, we conducted drug sensitivity and resistance testing (DSRT)  with 279TIMM23 and RP11-368 J21.2.1 in ZR-75-1 Tam1 and Tam2, and TNS1, PTH2R and NHLRC2 in BT-474 Tam1 and Tam2. None of the point mutations were shared between T-47D Tam1 and Tam2  and 0 (some ZR-75-1 and BT-474 networks), and converged into a number of target genes. In all but two networks (BT-474 Tam2 and ZR-75-1 Tam2 sensitivity) all the observed CNV changes were heterozygous deletions Figs. . Of the JAK2, possibly explaining the developing sensitivity towards gandotinib , but also several mTOR- and two HDAC-inhibitors   exhibited gained sensitivity towards the ERK1/2 inhibitor VX-11E. ERK1/2 inhibition prevents its autophosphorylation , 51 and We also identified bortezomib, a proteasome inhibitor, as a sensitizing agent Figs. , Table 1In addition to shared sensitivity to VX-11E and bortezomib in the tamoxifen-resistant cells, we also identified cell line specific drug sensitivities Table . T-47D TRAF1 promoting tamoxifen-resistant growth [Upon acquiring resistance to tamoxifen, the T-47D Tam1 cells also gained sensitivity towards the BCL-family inhibitors navitoclax and obatoclax Fig. . BCL-2 ft growth . Both nat growth –58 suggeWe also identified sensitizers with preference for the luminal B-derived tamoxifen-resistant cells, BT-474 Tam1 and Tam2. These included the cdk-inhibitors SNS-032 and alvocidib, along with the EGFR-inhibitor gefinitib and the Btk-inhibitor PCI-32765, and several HER2/EGFR-inhibitors Figs.  indicateDevelopment of primary drug resistance in cancer treatment frequently results in the emergence of secondary resistances. Here, we discovered that upon acquiring tamoxifen resistance, all of the resistant cells acquired co-resistance towards at least one chemotherapeutic agent, such as paclitaxel, docetaxel, vincristine, vinblastine or topotecan Figs.  and 12. BIRC5 has been shown to enhance cell sensitivity to tamoxifen [SLC35F2 gene [Interestingly, every single tamoxifen-resistant cell line was also more resistant to the survivin BIRC5-) inhibitor YM155 than their parental counterparts , and their cell biological, genomic and drug response profiling for future studies aiming to test novel therapies for breast cancer with acquired tamoxifen resistance.ER, estrogen receptor; DSRT, drug sensitivity and resistance testing; DSS, drug sensitivity score; KIBA–score, kinase inhibitor bioactivity –score; Ki, inhibitory constant; Kd, dissociation constant; IC50, half maximal inhibitory concentration; IPA, ingenuity pathway analysis; CNV, copy number variation; cdk, cyclin-dependent kinases


Listeria monocytogenes (LM). Extensive research in recent years has revealed that LLO exerts a wide array of biological activities, during the infection by LM or by itself as recombinant antigen. The spectrum of biological activities induced by LLO includes cytotoxicity, apoptosis induction, endoplasmic reticulum stress response, modulation of gene expression, intracellular calcium oscillations, and proinflammatory activity. In addition, LLO is a highly immunogenic toxin and the major target for innate and adaptive immune responses in different animal models and humans. Recently, the crystal structure of LLO has been published in detail. Here, we review the structure-function relationship for this fascinating microbial molecule, highlighting the potential uses of LLO in the fields of biomedicine and biotechnology, particularly in vaccination.Listeriolysin O (LLO) is a thiol-activated cholesterol-dependent pore-forming toxin and the major virulence factor of  Recentl-helices . The sig in vivo , 5, so iβ-strands and is connected to D4 through a glycine linker [d genetic background (BALB/c mice strain) [D2 is a key sequence to connect D1 to D4. D2 consists in four e linker , at resie linker . The imm strain) .β-sheet, which is surrounded by six α-helices [β-sheet with D320 and K316 from the second membrane-inserting helix bundle [β-sheets, which are organized forming a β-sandwich structure [D3 is formed by a five-stranded antiparallel -helices . Previou-helices . The recx bundle . The pH x bundle . D4 is tIt has been described that LLO is highly lytic for the nucleated cells and it can induce a wide range of different cell death types –23. The  in vitro results of apoptosis induction mediated by LLO are in agreement with in vivo observations. Injection of purified LLO into the footpads of mice led to TUNEL positive cells in the peripheral cortex and paracortex of the draining popliteal lymph node but not the more distal inguinal lymph node, confirming the proapoptotic activity of LLO in vivo [As a purified or recombinant protein, LLO induces cellular apoptosis in some primary immune cells, such as bone marrow dendritic cells (BMDC), BMM , primary in vivo .α-subunit of eukaryotic translation initiation factor-2, increase of the spliced X-box binding protein-1, and high expression of immunoglobulin binding protein [Recently, it has been described that LLO is able to induce other cellular activation pathways culminating in apoptosis induction. During LM infection in the P388D1 cell line, the bacterium induces the expansion of the endoplasmic reticulum (ER) and initiates a stress response to unfolded proteins (unfolded protein response or UPR). Induction of ER stress response is dependent on the production of LLO. LLO-deficient LM (LMΔhly) cells are not able to induce UPR. P388D1 cells stimulated with recombinant LLO reproduce UPR. Some activation markers of UPR increase after cell treatment with LLO or LM infection, such as the expression of protein disulfide isomerase, the processed form of activating transcription factor 6, phosphorylation of the  protein .The immune system is the collection of tissues, cells, and molecules that protects the body from numerous pathogenic microbes and toxins in our environment. The defense against microbes and microbial molecules includes two general types of responses: innate immunity and adaptive immunity. The innate immune system consists of diverse mechanisms, tissues, cells, and proteins that are always present and ready to mobilize and fight against pathogens and microbial molecules at the site of infection. The main components of the innate immune system are physical epithelial barriers, granulocytes such as neutrophils, eosinophils, mast cells, monocytes, and antigen presenting cells such as macrophages and dendritic cells (DCs), natural killer cells (NK cells), and circulating plasma proteins . The innα, IL-1β, IFN-γ, TNF-α, IL-6, iNOS, IL-10, and IL-12.In addition to the detrimental cytolytic and proapoptotic activities, LLO has been described as PAMP, which is recognized by different innate immune cells, such as peritoneal macrophages , BMM 2828, murinβ production in human PBMCs. Also the stimulation of human PBMCs with purified LLO induces the IL-1β secretion. Both infection with LM and stimulation with LLO induce the activation of a NOD-like receptor member: NLRP3, the best characterized inflammasome family member, which is critical for IL-1β production by PBMCs [β production [It has been shown recently that wild type LM, but not LM lacking LLO or expressing a non-pore-forming LLO, stimulates a strong IL-1by PBMCs . Interesoduction . Also, toduction .Different mechanisms are involved in the inflammatory response induced by LLO. The role of LLO as a toll-like receptor 4 (TLR-4) agonist is controversial. BMM from C3H/HeJ mice , do not respond with proinflammatory cytokine gene upregulation after stimulus with LLO . In cont2+ elevation in BMMCs in response to LLO has been described. Pretreatment of LLO with cholesterol inhibits the Ca2 influx, suggesting that this phenomenon is pore-forming dependent. Calcium influx induces degranulation, activation, and release of TNF-α. The TNF-α production involves the translocation of the nuclear factor of activated T-cells (NFAT), a key transcription factor for gene expression [Other activation mechanisms described for LLO include intracellular calcium oscillations. A cytosolic Capression .Another mechanism that may explain the cellular activation induced by LLO in murine macrophages is the aggregation of lipid rafts. Rafts aggregation is dependent of oligomerization of LLO. This cellular event induces tyrosine phosphorylation events and the accumulation of surface molecules such as CD14, a protein anchored to the membrane by a glycosylphosphatidylinositol tail and coreceptor along with TLR4 for the detection of bacterial lipopolysaccharide (LPS) .Adaptive immunity is mediated by antigen-specific immune mechanisms. The adaptive response and its specificity may be acquired following to the exposure to antigens, after the onset of infectious disease, by asymptomatic carriage of the pathogen, by harboring an organism with a similar structure (cross-reacting) or by vaccination. The white blood cells responsible for adaptive immunity include two different lymphocytes: T cells and B cells. Typically, B cells mediate humoral responses through the antibodies production. The main biological functions of antibodies are neutralization, complement activation, cellular cytotoxicity antibody dependent, and opsonization. Antibodies protect the host mainly against extracellular antigens. In contrast, T cells mediate cellular immune responses and recognize foreign antigen only when presented by major histocompatibility complex (MHC) molecules on the cell surface of antigen presenting cells (APCs) , 39. H-2 loci. H-2 is a complex of genetic loci on chromosome 17 of the mouse. Allelic differences in the H-2 complex affect host resistance to infection and cellular immune responses against specific antigens [In addition to the innate immune mechanism activated by LLO, the toxin is a prominent and primary target protein of the host's acquired immune system , 41. In antigens . in vivo against LM infection [For example, during the primary infection of BALB/c mice by LM, the cellular response is detectable between day 2 and day 4 after infection and reaches a peak production on day 6 . The adanfection .CD8+ T cell response is mainly focused on the immunodominant epitope LLO 91−99 produced in infected H-2d mice . In contThe infection of C57BL/6 mice with LM induces a strong CD4+ T cell response with an elevated frequency of LLO-specific cells but a poor anti-LLO CD8+ T cell response.In this particular genetic background, the immunodominant peptide for CD4+T cells includes the LLO 190–201 amino acids. Interestingly, the CD4+ T cell response against this antigenic region is also shared and conserved in BALB/c mice. Moreover, the CD8+ T cell response is directed to the 296–304 amino acid sequence . Similar in vivo cellular response is observed after immunization with wild type LLO into the footpads of C57BL/6 mice [ In vitro, the processing and presentation of LLO by murine APCs occur very rapidly. During the first 15–30 minutes, LLO is bound, internalized, and presented by APCs to induce the proliferation of LLO specific CD4+ and CD8+ T cell hybridomas. The presentation and activation of CD4+ T cell response is highly efficient, even at very low concentrations of LLO, such as the picomolar or femtomolar range [As a recombinant antigen, LLO is also a highly immunogenic molecule. A very robustL/6 mice . In vitrar range . A summaIn experimental animal models, LLO has been evaluated as an adjuvant in vaccination to induce protection against pathogens, allergies, and tumors. Administration of killed LM together with purified LLO induces protective immunity against the bacteria in mice . LLO alsγ, confirming LLO as the major contributor to induce this cytokine in immunized mice [γ. The intranasal challenge with recombinant LLO, in combination with OVA, suppressed the allergic responses, reduced the anti-OVA IgE titers in serum and the eosinophil infiltration in the nasal mucosa [p60 is a 60-kDa extracellular protein produced by LM and acts as a murein hydrolase required in the last step of cell division . Recombized mice . In an ol mucosa . Using al mucosa .LLO has also been used in the treatment of other experimental tumor models, such as follicular lymphoma and head and neck cancers. A chemically conjugated LLO with the 38C13 lymphoma Id protein (38Id-LLO) was evaluated as a vaccine and compared with the same antigen conjugated to keyhole limpet hemocyanin (KLH). 38Id-LLO induced very potent humoral and cellular responses and was very effective in inducing antilymphoma protection in immunized mice. Mice previously vaccinated with 38Id-LLO survived after a lymphoma challenge. As in the previously described models, conjugation to LLO polarized the T cell responses toward a Th1 profile, promoting a high titer of IgG2a anti-idiotype antibodies . In anotOther scientific evidence of LLO as a carrier/adjuvant molecule comes from recent studies with different model antigens. LLO can enhance the internalization, processing, and presentation of immunodominant peptides from hen-egg white lysozyme (HEL). A chimeric fusion protein that contains the 45–65 peptide of HEL protein fused to the aminoterminus region of LLO enhances by approximately 1000-fold the efficiency of presentation of the peptide to HEL-specific CD4+ T cell hybridomas, compared to the peptide alone . Another in vitro and in vivo biological systems, different technical approaches have been described for truncating or mutagenizing this molecule and studying its biological functions in the absence of its cytotoxic activity. Some examples of LLO mutants generated by different research groups and the resulting effects on its biological activities are shown in Given that LLO is a very potent toxin with adverse effects in bothBased on this evidence, it is clear that the LLO property of being a key target of the host innate immune response (PAMP molecule) is independent of its cytolytic activity. in vitro the IFN-γ production by mouse spleen cells. These results confirm that the key region for the innate immune recognition and the induction of proinflammatory cytokines is located in domains 1–3, in contrast to the role of domain 4 in binding to cholesterol membranes. Moreover, pretreatment of LLO with free cholesterol does not affect inflammatory activity but reduces the cytotoxicity, suggesting that the pore-forming property of cellular membranes is dispensable for the inflammatory induction mediated by LLO [Kohda et al. previously described that truncated forms of LLO, including domains 1–3 but not domain 4, are capable of inducingd by LLO .−/− deficient mice [A detoxified, nonhemolytic form of LLO (dtLLO) has been described recently , as an eent mice . Immunizent mice .Carrero et al. have shown that mutation of the tryptophans to alanines at both residues 491 and 492 (LLO W491-492A) or at only residue 492 (LLOW492A) led to a reduction in hemolytic activity of ~95–99.5% and in cytolytic activity to nucleated cells. Interestingly, the significant reduction in terms of cytotoxicity is not accompanied by a significant reduction in the uptake (~15–20%), binding, catabolism, processing, and presentation of immunodominant peptides to CD4+ or CD8+ T cells from these LLO mutants, confirming that antigenicity of LLO is also independent of its cytotoxic property . in vitro or in vivo by LLO, there are two key properties: first its proinflammatory capacity and second its potential for delivering antigens toward specific intracellular compartments in APCs.Vaccination is the most efficacious and valuable tool in the prevention of infectious diseases, and also it could be an important strategy for immunomodulation of some chronic pathologies such as allergies, autoimmunity, and cancer. Vaccines require optimal adjuvants and carrier molecules to achieve effective long term protection. With the knowledge gained in recent years about the structure, biological activities, and immune response to LLO, it has opened a potential and attractive use of this molecule in the applied research field. Of all the biological activities demonstratedThe ability of LLO fusion proteins to deliver antigens toward endosomal and cytosolic compartments for presentation by both MHC class I and class II molecules is an attractive approach to use LLO as an effective carrier molecule in vaccination. As a recombinant antigen LLO releases endosomal contents into the cytosol, providing a possible mechanism for entry of endocytosed antigens into the MHC class I pathway .In looking for safer molecules without the detrimental effects of LLO wild type, different technical approaches have generated multiple LLO-derived toxoids. Some important lessons we have learned from the manipulation of LLO structure are close to being applied in the vaccination field. First, now we know that for some LLO mutants the proinflammatory capacity is independent of its cytolytic activity , 29. Thi


The present study examined the expression of glucose-regulated protein 78 (GRP78/Bip) in human pancreatic cancer cell lines and the effect of knockdown of GRP78 on the cleavage of poly(ADP-ribose) polymerase (PARP). Human pancreatic cancer cell lines , constitutively expressed GRP78. We also demonstrated that ER stress induced by thapsigargin upregulated protein levels of GRP78. In the presence of thapsigargin, knockdown of GRP78 enhanced the PARP cleavage in the human pancreatic cancer cells. These results provide evidence that GRP78 is a potential therapeutic target for ‘difficult-to-treat’ pancreatic cancer, in which ER stress signaling in part falls into disorder. Pancreatic cancer is almost the deadliest of all malignancies . In JapaEndoplasmic reticulum (ER) stress response in tumor cells is critical for tumor cell growth and cancer progression . The ER The association between ER stress response and tumor growth and progression has been reported . We and We previously demonstrated that blocking of the induction of UPR, as well as inhibition of GRP78 expression is associated with the cleavage of poly(ADP-ribose) polymerase (PARP) . In the 2. Inhibitor of sarcoplasmic/endoplasmic reticulum (ER) Ca2+ ATPases (SERCA), thapsigargin, control siRNA (si-control) and siRNA for GRP78 (si-GRP78) were purchased from BioVision  and Santa Cruz Biotechnology , respectively.Human pancreatic cancer cell lines  were grown in RPMI-1640 medium  supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified atmosphere with 5% COTwenty-four hours after thapsigargin (1 μM) treatment, cells were lysed in sodium dodecyl sulfate sample buffer, and after sonication, lysates were processed for western blot analysis . BrieflyTo confirm the effects of GRP78 knockdown on apoptosis, we examined GRP78 knockdown by small-interfering RNA (siRNA). Cells were transfected with 50 nM si-GRP78 or si-control, using Effectene transfection reagent  according to the manufacturer’s protocol . After 2Results are expressed as means ± standard deviation (SD). Statistical analysis was performed using the Student’s t-test. A P-value <0.05 was considered to indicate a statistically significant result.First, we examined the GRP78 expression in the human pancreatic cancer cell lines SUIT-2, MIAPaCa-2, Panc-1 and KP-2 . ProteinNext, we examined the effect of thapsigargin, one of the ER stress-inducers, on GRP78 expression in the human pancreatic cancer cell lines . TreatmeWe examined the protein expression of ER stress signaling-associated molecules in the human pancreatic cell lines treated with or without thapsigargin. The results for the Panc-1 and KP-2 cells are shown in On the other hand, in KP-2 cells, the protein expression levels of GADD34, ATF4, ATF6 and XBP1 were upregulated in the presence of 1 μM thapsigargin  .XBP1 was also upregulated in the presence of 1 μM thapsigargin in both SUIT-2 and MIAPaCa-2 cells, yet we did not observe any enhancement of GADD34, ATF4 or ATF6 by thapsigargin (data not shown).We also examined the phosphorylation status of eIF2α to understand how thapsigargin affects ER stress signaling in Panc-1 and KP-2 cells . In PancWe confirmed that the expression of GRP78 at the protein level was upregulated in all four human pancreatic cancer cell lines tested, yet other molecules downstream of GRP78 reported to be involved in ER stress were expressed at variable levels depending on the individual cell line. Thus, we focused our examination on GRP78. Our previous study  demonstrGRP78 expression was significantly inhibited by transfection with si-GRP78 in the presence of thapsigargin, compared with that with si-control [1.4±0.040 vs. 1.8±0.040, n=3, p=0.00014; and 7.1±0.24 vs. 18.3±0.37, n=3, p=0.0000038, respectively, in Panc-1  and MIAPPARP cleavage was significantly enhanced by transfection with si-GRP78 in the presence of thapsigargin, compared with that with si-control [4.5±0.045 vs. 1.6±0.085, n=3, p=0.00000080; and 2.6±0.13 vs. 1.5±0.047, n=3, p=0.00016, respectively, in Panc-1  and MIAPIn the present study, we demonstrated that i) human pancreatic cancer cell lines expressed GRP78; ii) ER stress induced by thapsigargin upregulated protein levels of GRP78 in human pancreatic cancer cell lines; iii) ER stress-related molecules downstream of GRP78 were expressed at various levels according to the respective human pancreatic cancer cell lines; and iv) finally, knockdown of GRP78 by siRNA enhanced PARP cleavage in the human pancreatic cancer cell lines. To our knowledge, this is the first report to show the association between GRP78 and PARP cleavage in pancreatic cancer cell lines treated with thapsigargin.Our results that human pancreatic cancer cell lines express GRP78 supported a previous study  showing It was reported that an increase in GRP78 expression in pancreatic cancer cells may enhance and account for the altered sensitivity of pancreatic cancer to chemotherapeutic agents . UPR reget al reported that suppression of GRP78 by taxol and vinblastine potentiated the activation of JNK phosphorylation, caspase-7 and PARP cleavage in the human breast cancer cell line MCF-7 (PARP is one of the proteins processed by post-translational modification and plays a crucial role in many processes, including DNA repair and cell death . During ne MCF-7 . The Hspne MCF-7 .Collectively, our results suggest that both GRP78 and PARP may have key roles in the chemoresistance of pancreatic cancer  and that


L are associated with significant thrombocytopenia. This has led to the development of ABT-199 which specifically inhibits Bcl-2. Unfortunately, Bcl-XL appears to be more frequently deregulated in ovarian cancer than Bcl-2. We therefore compared the ability of ABT-199, and the Bcl-XL selective compound WEHI-539, to potentiate the activity of carboplatin in ovarian cancer cell lines.BH3 mimetics are a class of drugs that antagonize the Bcl-2 family of apoptosis inhibitors. We have previously shown that these compounds can potentiate the activity of carboplatin against several ovarian cancer cell lines. However, recent clinical studies have highlighted that BH3 mimetics which antagonise Bcl-XWEHI-539, ABT-737 and ABT-199 were tested in combination with carboplatin using a panel of 6 ovarian cancer cell lines. The activity of the drugs was evaluated using cell growth assays, staining with trypan bue and measurement of apoptosis by measuring caspase 3/7 activity, PARP cleavage and annexin-V/propidium iodide staining.We found that WEHI-539 and ABT-737, but not ABT-199, were synergistic with carboplatin in cell growth assays and potentiated cell death when assessed by trypan blue staining. Furthermore, WEHI-539 and ABT-737 augmented carboplatin induced caspase 3/7 activity, PARP cleavage and annexin V labelling, but ABT-199 failed to do so.L are necessary if BH3 mimetics are to be successfully used to treat patients with ovarian cancer and this highlights the need to develop strategies to minimize thrombocytopenia induced by such compounds.These observations suggest that compounds which target Bcl-X Ovarian cancer (OC) is a heterogeneous disease characterized by low incidence, affecting around 4 % of women, but with rapid progression and high mortality rate . AlthougL, Bcl-W, Mcl-1, and Bcl-2A1 act as inhibitors of this pathway by sequestering other pro-apoptotic family members [L and Bcl-W but not Mcl-1 [L in platelets [L. Clinical studies have demonstrated that ABT-199 does not cause significant thrombocytopenia and its efficacy is currently being evaluated in a number of cancer types [L (14 %), Bcl-W (12 %), or Mcl-1 (14 %) is observed is notably higher. This led us to question whether a Bcl-2 selective inhibitor would be of therapeutic use in a significant proportion of ovarian cancer patients. Instead, we hypothesized that a Bcl-XL selective inhibitor would be preferable, although additional strategies would be necessary to overcome the likely ensuing thrombocytopenia. WEHI-539 is a recently described selective inhibitor of Bcl-XL. We therefore compared the ability of ABT-199 and WEHI-539 to potentiate the activity of carboplatin. ABT-737, which antagonises Bcl-2, Bcl-XL and Bcl-2, was included as a comparator. In several models, we found that WEHI-539, as well as ABT-737, augmented the activity of carboplatin but ABT-199 failed to do so. These observations suggest that compounds targeting Bcl-XL will be of benefit in ovarian cancer, but novel strategies to minimize thrombocytopenia will be necessary.One of the major causes for the development of drug resistance is faulty apoptosis, one cause of which is overexpression of anti-apoptotic members of Bcl-2 family . The con members –9. BH3-m members . In addi members . Autophaot Mcl-1 , 13. We ot Mcl-1 , 15. Altlatelets . As a reer types –19. Thiser types . This iser types . In contOvcar-8, Ovcar-3, Ovcar-4 and Ovsaho cells were grown in the RPMI, Igrov-1, Cov-362 and Cov-318 cells were grown in DMEM and Fuov-1 cells were grown in DMEM/F-12 nutrient mixture. Growth medium were supplemented with 10 % FCS, L-Glutamine (2 mM) and penicillin-streptomycin (50 IU/ml). For Ovcar-3, cells the medium was further supplemented with Insulin (0.01 mg/ml) and sodium pyruvate (0.11 g/l). ABT-737, ABT-199 and WEHI-539 , were prepared as a 20 mmol/L solution in dimethyl sulfoxide, whereas the stock solution of carboplatin was prepared in phosphate-buffered saline (PBS) at 13.5 mmol/L.For cell growth assays, cells were plated in 96 wells plate . The next day, cells were treated with drugs. After 72 h the culture medium was removed and the cells were fixed with 100 μl of cold 10 % Trichloroacetic acid (TCA), incubated on ice for 30 min and stained with 0.4 % sulforhodamine B (SRB) as described . The datTo assess viability using trypan blue, cells were plated in 12 well plates and exposed to the indicated drug concentration for 48 h except for Ovcar-4 which was assessed after 24 h. The supernatant was collected and adherent cells detached with trypsin. The two samples were combined, and cells collected by centrifugation  before re-suspending in 0.2 % trypan blue in PBS and counting live and dead cells on a haemocytometer.To quantify apoptosis, cells were plated in 96 well plates and exposed to the indicated concentration of carboplatin and/or BH3 mimetic as described above. After 48 h, 25 μl of Caspase 3/7 Glo-reagent (Promega) was added and luminescence measured after 30 min. The Bliss independence criterion , 25 was Where E is the expected effect of the combination and E(x) and E(y) are the effect of individual drugs.To measure PARP cleavage, cells were seeded in 12 wells plate and treated with drugs and collected as described above. After washing with PBS, cells pellets were lysed with RIPA buffer (3 ml) comprising 20 mM Hepes, 150 mM NaCl, 2 mM ethylenediamine tetra-acetic acid, 0.5 % sodium deoxycholate, 1 % Nonidet-P40, and supplemented with 0.12 mM leupeptin, 0.05 mM pepstatin and 1 mM phenylmethylsulfonyl fluoride. Proteins concentration was estimated with a bicinchoninic acid (BCA) assay. Samples (10 μg) were analysed on 4-20 % Hepes gels (NuSep), electro-transferred to polyvinyldifluoride and analysed with PARP antibody .To measure annexin V labelling, cells were seeded in 12 well plates, exposed to the indicated drug concentration for 48 h. The cells were labelled using a Annexin V-FITC kit (Miltenyi biotech) according to the manufactur’s instructions and analysed by flow cytometry.L was expressed in all 8 cell lines. Bcl-2 expression was more variable, although it was detectable in 5 lines it was only prominent in 3 cell lines. Mcl-1 expression was also notable in 5 cell lines. In cell growth assays, all three compounds inhibited the growth of cultures of each cell line with microMolar potencies  was synergistic with carboplatin in three cell lines (Fig. L selective compound WEHI-539 was also synergistic with carboplatin in these cell lines. In Ovcar-4 and Ovsaho, synergy between ABT-737 and carboplatin was not observed. In contrast, the Bcl-2 selective compound ABT-199 was not synergistic with carboplatin in any of the cell lines and was even antagonistic with carboplatin in Ovcar-8, Igrov-1 and Ovsaho cells.To evaluate which of the BH3 mimetics was synergistic with carboplatin, drug combination studies were performed in 6 cell lines using a fixed concentration of each BH3 mimetic and a range of concentrations of carboplatin. ABT-737 (which inhibits Bcl-2 as well as Bcl-XL compared to Mcl-1 (Fig. To confirm these results, the effects of the drug combinations on cell viability were assessed using trypan blue staining Fig. . ConsistL (Fig. In addition to the effect in cell growth assays, we have previously shown that ABT-737 and navitoclax potentiate apoptosis induced by carboplatin. To investigate the effect of the selective BH3 mimetics we measured caspase activity, PARP cleavage and Annexin V/Propidium iodide staining. The activation of caspase 3/7 by the drug combinations Fig.  was remiL Fig.  and in wL Fig. , the effL (Fig. L compared to MCl-1 (Fig. In Ovcar-3, Ovcar-8, Igrov and Cov-362 cells, the BH3 mimetics did not induce noticeable PARP cleavage when they were tested as single agents Fig. . As a siL Fig. . ABT-737l-1 Fig. . In contFlow cytometry experiments assessing annexin V/propidium iodide staining Fig.  showed tL, potentiate the activity of carboplatin in ovarian cancer cell lines [L selective compounds ABT-199 and WEHI-539 to potentiate the activity of carboplatin. WEHI-539 was synergistic with carboplatin in several cell lines using several assays including cell growth, cell death, activation of caspase 3/7, PARP cleavage and Annexin V/propidium iodide staining. In contrast, in the same set of assays, ABT-199 did not potentiate the cell death induced by carboplatin and was even mildly antagonistic with carboplatin in some assays.Dysregulation of apoptosis is regarded as one of the major contributors to drug resistance, which may result from overexpression of the anti-apoptotic Bcl-2 proteins , 28. Tarll lines , 15. In The observation that ABT-199 is antagonistic with carboplatin in several ovarian cancer cell lines is concerning because this compound is undergoing clinical evaluation as an alternative to navitoclax, a BH3 mimetic pharmacologically similar to ABT-737 and which causes thrombocytopenia. The reason for antagonism between carboplatin and ABT-199 that was observed in several cell lines is unclear. Antagonism was most evident in Ovcar-3, Ovcar-8 and Igrov-1 cells which express relatively low amounts of Bcl-2. Additivity was observed between carboplatin and ABT-199 and carboplatin in Ovcar-4 cells and COV-362 cells which expressed higher levels of Bcl-2. Bcl-2 has been linked to regulation of the cell cycle and DNA damage responses , 30 and L, which is inhibited by both ABT-737 and WEHI-539. It is also noteable that in this cell line ABT-737 and WEHI-539 induced apoptosis when they were tested as single agents. Thus, the lack of synergy between carboplatin and these compounds in Ovcar-4 cells may reflect the low level of Bcl-XL expression which allows these BH3 mimetics to induce apoptosis on their own, so synergy is not readily observed.Synergy between ABT-737 or WEHI-539 and carboplatin was observed in several cell lines using several different assays. The pre-eminent exception to this was Ovcar-4 cells, where the combinations of these were additive in all the cell lines we tested. It is noteable that, of all the cell lines evaluated, Ovcar-4 express the lowerst level of Bcl-XL will be necessary if BH3 mimetics are to be successfully used in the treatment of ovarian cancer. This is reinforced by the more frequent deregulation of Bcl-XL than Bcl-2 in ovarian cancer reported in the cancer genome atlas [L and Bcl-w and consequently it is likely to be effective in potentiating the activity of carboplatin in a larger proportion of ovarian cancer patients than ABT-199. However, navitoclax has been closely linked to thrombocytopenia. The same may be anticipated with WEHI-539 because both drugs inhibit Bcl-XL that promotes platelet survival. A therapeutic window may be found by careful dose escalation studies, Alternatively, novel strategies may be necessary to minimize this adverse effect. For example, encapsulation of ABT-737 in nanoparticles has been shown to enhance its anti-tumour effects while minimizing thrombocytopenia [Our data strongly support the argument that drugs which inhibit Bcl-Xme atlas . Consequme atlas . A persoytopenia . Alternaytopenia . This maWe conclude that navitoclax remains the preferred BH3 mimetic for the treatment of ovarian cancer, but strategies to minimize thrombocytopenia will be essential if this drug is to be used safely.The datasets supporting the conclusions of this article are included within the article.


Human cancers frequently arise from increased expression of proto-oncogenes, such as MYC and HER2. Understanding the cellular pathways regulating the transcription and expression of proto-oncogenes is important for targeted therapies for cancer treatment. Adenoviral (Ad) E1A 243R (243 aa residues) is a viral oncoprotein that interacts with key regulators of gene transcription and cell proliferation. We have shown previously that the 80 amino acid N-terminal transcriptional repression domain of E1A 243R (E1A 1-80) can target the histone acetyltransferase (HAT) p300 and repress HER2 in the HER2-overexpressing human breast cancer cell line SKBR3. Expression of E1A 1-80 induces death of SKBR3 and other cancer cell lines. In this study, we performed total cell RNA sequence analysis and identified MYC as the regulatory gene for cellular proliferation most strongly repressed by E1A 1-80. By RT-quantitative PCR analysis we show that repression of MYC in SKBR3 cells occurs early after expression of E1A 1-80, suggesting that MYC may be an early responder of E1A 1-80-mediated transcriptional repression. Of interest, while E1A 1-80 repression of MYC occurs in all eight human cancer cell lines examined, repression of HER2 is cell-type dependent. We demonstrate by ChIP analysis that MYC transcriptional repression by E1A 1-80 is associated with inhibition of acetylation of H3K18 and H4K16 on the MYC promoter, as well as inhibition of RNA Pol II binding to the MYC promoter. Deletion mutant analysis of E1A 1-80 suggests that both p300/CBP and TRRAP are involved in E1A 1-80 repression of MYC transcription. Further, E1A 1-80 interaction with p300/CBP and TRRAP is correlated with inhibition of H3K18 and H4K16 acetylation on the MYC promoter, respectively. Our results indicate that E1A 1-80 may target two important pathways for histone modification to repress transcription in human cancer cells. Ad E1A 243R (243 amino acid residues) is a viral oncogene product that intimately interacts with cell cycle regulatory pathways to establish a favorable environment for viral DNA replication. E1A functions as a master regulator of chromatin remodeling and promoter activity by targeting the histone acetyltransferase (HAT) p300/CBP and TRRAP through the N-terminal region and conserved region 1 (CR1), tumor suppressor Rb through CR2, and transcriptional co-repressor CtBP through C-terminal CR4 . NeWe have reported that SKBR3 cells, a human breast cancer cell line that over-expresses HER2, are efficiently killed by expression of E1A 1-80 [To examine if E1A 1-80 regulates transcription of HER2 and EGFR in other breast cancer cell lines, MCF7 and MB231 cells were infected with Ad-lacZ or Ad-E1A 1-80 C+, and RT-qPCR analysis performed. E1A 1-80 C+ represses transcription of HER2 but not EGFR in MCF7 cells Figure , whereasTo further understand transcriptional repression by E1A 1-80, we performed total cell RNA-seq analysis with triplicate RNA samples prepared from SKBR3 cells infected with Ad-lacZ or Ad-E1A 1-80 C+. RNA-seq analysis with oligo-dT-selected mRNA revealed a number of genes whose expression is modulated by E1A 1-80 C+ see . We seleE1A 1-80 repression of the selected genes in SKBR3 cells was confirmed by RT-qPCR analysis, and further examined in two additional human breast cancer cells lines MB231 and MCF7 that are also efficiently killed by E1A 1-80 C+ . CCND1 iAmong the genes analyzed, MYC is the most strongly repressed by E1A 1-80 C+ in the breast cancer cell lines tested. To examine if MYC is efficiently repressed by E1A 1-80 C+ in cell lines derived from different cancer types, additional human cancer cell lines were infected with Ad-lacZ or Ad-E1A 1-80 C+, and RT-qPCR was performed. Among the cell lines chosen, MB468 is derived from a breast cancer, HeLa from a cervical cancer, A549 from a lung cancer, HCT116 from a colon cancer, and U2OS from an osteosarcoma. MYC is efficiently repressed by E1A 1-80 C+ in all cell lines tested Figure . BCL3 isin vivo and in vitro on assembled chromatin [in vivo, SKBR3 cells were infected with Ad-lacZ or Ad-E1A 1-80 C+, fixed with formaldehyde and processed for ChIP analysis as described in Figure It was previously reported that E1A 243R inhibits global H3K18 acetylation, an epigenetic mark catalyzed by p300/CBP . We haveAssembly of RNA Pol II on the promoter is a prerequisite for gene transcription. We therefore performed ChIP assay with Pol II antibody. Pol II antibody enriched MYC promoter by less than 0.2% Figure  in cellsAcetylation of H4K16 is associated with transcriptionally active genes in mouse embryonic stem cells and is independent of p300/CBP activity . H4K16 aAs negative control, chromatin from cells infected with Ad-lacZ was immunoprecipitated with non-specific mouse or rabbit IgG, and the ChIP DNAs examined for the presence of MYC, HER2, and CTRL by qPCR Figure . As showThese combined results show that E1A 1-80 C+ strongly inhibits H3K18 acetylation and Pol II binding to the MYC promoter in SKBR3 cells. By comparison, E1A 1-80 C+ inhibits H3K18 acetylation and Pol II binding to the HER2 promoter to a lesser extent. However, E1A 1-80 C+ represses H4K16 acetylation only on the MYC promoter .Human cancer cell lines SKBR3, MB231 (MB-MD231), MB468 (MB-MD468), MCF7, HeLa, HCT116, A549, and U2OS were cultured in DMEM supplemented with 4.5 g glucose/L, 5 mM glutamine, Pen/Strep, and 10% fetal bovine serum (Invitrogen) in a 37°C incubator with 5% COE1A 1-80 C+ Ad vector construction and preparation were as described previously . Ad-E1A 5 cells per well in 6-well plates  or 6 × 105 cells per T25 flask , and infected as described [For infection, cells were plated at a density of 3 × 10escribed , 51. qPC+-RNAs from the samples were enriched prior to library construction.Triplicate samples of SKBR3 cells were infected with Ad-lacZ or Ad-E1A 1-80 C+, and RNA prepared as described above. RNA-seq analysis was performed with the assistance from the Washington University Genome Technology Access Center. PolyA6 SKBR3 cells were plated in T175 flasks, and infected with Ad vectors the next day. ChIP assays were performed using the MAGnify ChIP assay kit (Invitrogen) following manufacturer's recommendations. Formaldehyde-fixed cells were sonicated for a total of 2 min with alternate 15 s pulse and 15 s rest, at 10% amplitude using a Branson Digital Sonifier (model 450) with a microtip. For ChIP immunoprecipitation, 3 μg of antibody (or 3 μl of anti-serum) and 200 μl of 1:10 diluted chromatin were used. Input DNA controls represent 1% of the chromatin used for ChIP. Following ChIP, qPCR was performed under the same conditions as for RT-qPCR , H3K18Ac: #07-354 (Millipore), RNA Pol II: #49-1033 (Invitrogen), E1A: rabbit polyclonal to E1A 1-80.


The aim of the study was to characterize a recurrent amplification at chromosomal region 1p21-22 in bladder cancer.ArrayCGH (aCGH) was performed to identify DNA copy number variations in 7 clinical samples and 6 bladder cancer cell lines. FISH was used to map the amplicon at 1p21-22 in the cell lines. Gene expression microarrays and qRT-PCR were used to study the expression of putative target genes in the region.TMED5, DR1, RPL5 and EVI5, showed significant overexpression in the SCaBER cell line compared to all the other samples tested. Oncomine database analysis revealed upregulation of DR1 in superficial and infiltrating bladder cancer samples, compared to normal bladder.aCGH identified an amplification at 1p21-22 in 10/13 (77%) samples. The minimal region of the amplification was mapped to a region of about 1 Mb in size, containing a total of 11 known genes. The highest amplification was found in SCaBER squamous cell carcinoma cell line. Four genes, DR1 was found to be significantly overexpressed in the SCaBER, which is a model of squamous cell carcinoma. However, the overexpression was found also in a published clinical sample cohort of superficial and infiltrating bladder cancers. Further studies with more clinical material are needed to investigate the role of the amplification at 1p21-22.In conclusions, we have identified and mapped chromosomal amplification at 1p21-22 in bladder cancer as well as studied the expression of the genes in the region.  Bladder cancer is the fourth most common cancer in men in developed countries and the second most common malignancy of the urinary tract . The majin situ hybridization (FISH), Southern blot analysis, quantitative polymerase chain reaction (PCR)-based assays and comparative genomic hybridization (CGH) have been used [Several studies have investigated the chromosomal alterations associated with development and progression of bladder cancer. Different methods to detect copy number changes, such as classical cytogenetics, interphase fluorescence een used .CDKN2A[DBCCR1[TSC1[PTEN locus [RB1[TP53[CCND1) at 11q13 [ERBB2 at 17q21 [E2F3 at 6p22 [MDM2 at 12q14 [MYC at 8q24 [HRAS[FGFR3[RAS and FGFR3 and loss-of-function mutation affecting RB, PTEN and TP53 have also been associated with the pathological stage and/or outcome of bladder cancer [Several CGH studies providing information about typical losses, gains and amplifications in bladder cancer have been published –8. HowevCDKN2A, DBCCR1[DBCCR1, and TSCR1[TSC1. DeletioEN locus , 20, 13qcus [RB1 and 17p B1[TP53. Common at 11q13 , 24, ERBat 17q21 , 26, E2F at 6p22 , 28, MDMat 12q14 , and MYC at 8q24 . Recurre24 [HRAS and FGFRS[FGFR3 seem to r cancer , 34.In this study, we report the characterization of a common amplification at chromosomal region 1p21-22. The amplicon was identified by aCGH analysis of clinical specimens obtained from bladder cancer patients and in bladder cancer cell lines.Freshly frozen samples from 7 bladder cancer tissues were used for this study. The samples were obtained from Tampere University Hospital and include five urothelial carcinomas, one lymphoepithelial carcinoma and one undifferentiated carcinoma. DNA was extracted using DNAzol reagent , according to manufacturer’s protocol. The use of the clinical samples was approved by the ethical committee of the Tampere University Hospital.The bladder cancer cell lines UM-UC-3, TCCSUP, RT4, T24, HT-1376, J82, SCaBER, 5637, HT-1197 and SW780 were obtained from the American Type Culture Collection  and cultured according to the recommended conditions.http://www.btk.fi/microarray-and-sequencing/) . The poly-L-lysine coated slides contain approximately 16000 annotated clones from sequence verified I.M.A.G.E. Consortium cDNA library in duplicate. Comparative genomic hybridization to microarray (aCGH) was done as described previously [16 K cDNA microarray-slides were obtained from the Finnish Microarray DNA Centre , according to the manufacturer’s protocol. The samples were then amplified and hybridized using the Agilent whole genome oligo microarray platform  and Xpress Ref ™ Human Universal Reference Total RNA (SuperArray Bioscience Corporation) was used as a reference. The resulting data files from Agilent Feature Extraction Software (version 9.5.1.1) were imported into the Agilent GeneSpring GX software (version 11.0) for further analysis. A fold-change cutoff of 2 was used to determine differential gene expression.Total RNA from bladder cancer cell lines, extracted as described above, was reverse transcribed using random hexamere primers and AMV reverse transcriptase (Thermo Scientific). Quantitative Real Time PCR was performed using Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific) and a BioRad CFX96 ™ Real-Time PCR Detection System. Each sample was run in duplicate and expression values were normalized against TATA-binding protein (TBP). The primer sequences are shown in Table The CLAC-analysis of the aCGH data from clinical samples and bladder cancer cell lines showed a region of increased copy number at chromosome 1p21-22 in 5 of 7 total clinical samples as well as in bladder cancer cell lines, 5637, RT4, T24, SW780 and SCaBER (data not shown). According to aCGH, the common region of gain comprised of 2 Mb.The region 1p21-22 was studied in bladder cancer cell lines by FISH analysis on interphase nuclei Figure .Figure 1DR1, EVI5, RPL5 and TMED5 only in the SCaBER, which harbors the highest level of amplification of the region  upregulation of the gene in clinical samples of both superficial and infiltrating bladder cancer, when compared to normal bladder [TMED5 showed significant upregulation in superficial bladder cancer, when compared to normal, whereas RPL5 and EVI5 did not show significant changes of expression levels in the same dataset.The analysis of gene expression by microarray showed significant overexpression of 4 genes, namely n Figure . The resn Figure . In addi bladder  (Figure In this study, aCGH technology was utilized to identify new regions of amplifications in bladder cancer. Recurrent amplification was found in chromosomal locus 1p21-22. Subsequently, the locus was fine-mapped and characterized in the bladder cancer cell lines. Of the cell lines SCaBER showed the highest amplification of the region, thus it was used for mapping the amplicon. Fine mapping with the SCaBER model, the region was defined to ~1 Mb of size, containing 11 genes.DR1, EVI5, RPL5, and TMED5 showed overexpression in SCaBER compared to the other cell lines. DR1 was found to be the most significantly overexpressed of the examined genes. Since SCaBER is a squamous cell carcinoma cell line, we wished to interrogate whether DR1 is overexpressed also in the urothelial carcinoma. We utilized Oncomine database of clinical samples, which showed overexpression of DR1 also in superficial and infiltrating bladder cancer.cDNA microarray and qRT-PCR analyses were used to measure the expression of these genes in bladder cancer cell lines. DR1 is also known as NC2beta and has been shown to bind DRAP1 to repress RNA polymerase II gene transcription [DR1/DRAP1 inhibition [cription . Despitehibition  and the hibition .DR1, EVI5, TMED5 and RPL5 are co-amplified also in brain [According to Oncomine database in brain –43, coloin brain , lung cain brain  and melain brain , indicatDR1. In a published data set, DR1 was also overexpressed in clinical samples of superficial and infiltrating bladder cancers, suggesting that DR1 is a putative target for the amplification. Further studies are needed to assess the role of the amplification at 1p21-22 in bladder cancer.We have identified and mapped a common chromosomal amplification at 1p21-22 in bladder cancer. Squamous cell carcinoma cell line SCaBER, which had the highest level of amplification of the region, showed overexpression of


The development of resistance against metronidazole , thrown a challenge to find out alternate medication.Trichomoniasis is the most common sexually transmitted To Design and synthesize novel agents to be effective against MTZ resistant trichomoniasis.Trichomonas.[Trichomonas activity.[14-28) as N-alkyl/aralkyl-4-(3-substituted-3-phenylpropyl)piperazine-1-carbodithioates have been designed, synthesized and evaluated for their anti-Trichomonas activity profile to be useful as vaginal microbicide. All compounds were tested for safety through cytotoxic assay against human cervical cell line (Hela) and compatibility with vaginal flora, Lactobacillus.Benzenepropanamines and selective serotonin reuptake inhibitor (SSRI) antidepressants viz. fluoxetine and paroxetine, possibly interacting with sulfhydryl groups present over chomonas. Alongsidactivity. In our oCompound 17) was the most promising compound with anti–Trichomonas activity  in comparison to MTZ . Six compounds  were more active against resistant strain in comparison to Metronidazole. The extreme safety profile against vaginal epithelium (HeLa cells) and compatibility with vaginal flora (lactobacillus) supported its suitability for vaginal application.2-(pyrrolidin-1-yl)ethyl 4-(3-oxo-3-phenylpropyl)piperazine-1-carbodithioate (A novel molecule to be effective against resistant Trichomoniasis in comparison to MTZ has been identified to be developed for topical application emphasizing on improvement of women reproductive health.None declared.


Zalypsis® is a marine compound in phase II clinical trials for multiple myeloma, cervical and endometrial cancer, and Ewing’s sarcoma. However, the determinants of the response to Zalypsis are not well known. The identification of biomarkers for Zalypsis activity would also contribute to broaden the spectrum of tumors by selecting those patients more likely to respond to this therapy.in vitro drug sensitivity data coupled with a set of molecular data from a panel of sarcoma cell lines, we developed molecular signatures that predict sensitivity to Zalypsis. We verified these results in culture and in vivo xenograft studies.Using in vivo in xenograft models. All cells lines expressing low levels of RTK signaling were sensitive to Zalypsis in vivo, whereas all cell lines except two with high levels of RTK signaling were resistant to the drug.Zalypsis resistance was dependent on the expression levels of PDGFRα or constitutive phosphorylation of c-Kit, indicating that the activation of tyrosine kinase receptors (TKRs) may determine resistance to Zalypsis. To validate our observation, we measured the levels of total and active (phosphorylated) forms of the RTKs PDGFRα/β, c-Kit, and EGFR in a new panel of diverse solid tumor cell lines and found that the IC50 to the drug correlated with RTK activation in this new panel. We further tested our predictions about Zalypsis determinants for response RTK activation might provide important signals to overcome the cytotoxicity of Zalypsis and should be taken into consideration in current and future clinical trials. During the past 30 years, medical oncologists have focused on optimizing the outcome of cancer patients by developing new antitumoral agents and defining new prognostic factors as well as integrating more effective supportive care measures. However, clinical anticancer strategies indicate that conceptually active therapies benefit only a small proportion of patients; thus, a large cohort of patients must be exposed to these antitumoral treatments to obtain a benefit in only a fraction of them.Pharmacogenomic studies are aimed at identifying predictive biomarkers that can help to define subpopulations of patients who will, or will not, benefit from a particular therapy. These molecular markers of a response to a specific drug are not exclusive to the so-called “Targeted Therapies” but also have been identified for widely used cytotoxic agents. Representative examples include the relationship between mRNA expression and response and survival using antifolates, beta tuin vitro and in vivo, including cell lines resistant to other chemotherapeutic agents[http://clinicaltrials.gov/ct2/show/NCT01222767).Zalypsis® (PM00104) is a marine-derived compound that has shown cytotoxic activity against various human tumor cell lines both c agents,6. ZalypIn vitro data suggests that Zalypsis has DNA-binding properties, induces cell cycle arrest, and inhibits transcription, eventually leading to apoptosis[Schizosaccharomyces pombe cells containing a RAD51 mutation were found to be extremely sensitive to Zalypsis, suggesting that the compound induces double-strand breaks (DSBs)[Structurally, Zalypsis contains a similar chemical scaffold to trabectedin, differing in an additional appended ring. It has poptosis,11. Althpoptosis,13. The s (DSBs). Howeverin vitro and in xenograft models in vivo.The aim of this study was to identify biomarkers defining the molecular basis of sensitivity/resistance to Zalypsis to assist in its clinical development. To this end, we used a panel of solid tumors, including low-passage cell lines from untreated sarcoma tumor samples. Using tThe panel was composed of commercial and in-house-generated cell lines from patients of soft tissue sarcomas. For the generation of new cell lines, sterile fragments from resected tumors were minced in culture medium and then disaggregated by 1–2 h incubation in collagenase (100 U/ml) at 37°C. After 24 h, the medium was changed to F-10 Ham (Gibco) supplemented with 1% Ultroser G (Biosepra). The cell lines generated were cultured in F-10 Ham supplemented with 1% Ultroser G. A673 cells were cultured in RPMI (Sigma) and SW872 in Leibovitz L-15 (Sigma). All media were supplemented with 10% FBS, fungizone, and penicillin/streptomycin. Once the cells became confluent, adherent cells were removed by trypsin treatment and seeded at 1/2 or 1/3 ratio with medium. Throughout the establishment of these cell lines, their phenotypic features were followed. Additionally, the cell lines were routinely checked for mycoplasma contamination (INVIVOGEN). All cell lines used were established immortal tumor cell lines.For the newly created human cell lines from resected tumor tissue, approval from local ethics committee at Hospital Universitario Virgen del Rocio  was obtained (PI2012-0085) and informed consent was obtained from patients.The compounds were tested using 96-well trays. Cells growing in a flask were harvested just before becoming confluent, counted using a hemocytometer, and diluted with media by adjusting the concentration to the required number of cells per 0.2 ml (volume for each well). The cells were then seeded in 96-well trays at a density between 1,000 and 4,000 cells/well, depending on the cell size. The cells were allowed to settle down and grow for 24 hours before adding the drugs, which were weighed and diluted with DMSO to a concentration of 10 mM. A “mother plate” with serial dilutions was prepared at 200X the final concentration in the culture; 11 different concentrations were tested at 1/3 dilution in a range from 10 μM to 0.1 nM. When necessary, for highly sensitive or highly resistant cell lines, a new mother plate was generated by decreasing or increasing two more concentrations, as required. The final concentration of DMSO in the tissue culture media did not exceed 0.5%. The appropriate volume of the compound solution  was added automatically (Beckman FX 96 tip) to the media to reach the final concentration for each drug. The medium was removed from the cells and replaced with 0.2 ml of medium dosed with drug. Each concentration was assayed in triplicate. Two sets of control wells were included in each plate, containing either medium without drug or medium with the same concentration of DMSO. A third control set was obtained with the untreated cells just before the addition of the drugs . The cells were exposed to the drugs for 96 hours and then washed twice with phosphate-buffered saline before being fixed with 10% glutaraldehyde. The cells were washed twice, fixed with crystal violet 0.5% for 30 minutes, washed extensively, solubilized with 15% acetic acid, and absorbance measured at 595 nm. The value of cytotoxicity was given as an IC50 concentration, the concentration a particular drug needed to inhibit by 50% the proliferation of a cell line or kill 50% of a cell population.Total RNA was collected using the TRI-REAGENT . RT was performed (Promega) with 1 μg of RNA following the manufacturer’s protocol. cDNA (1 μg) was used for PCR, and the amplified products were analyzed by electrophoresis on a 1% agarose gel. The PCR primers used and the length of the amplified product are shown in Additional fileWhole-cell extracts were prepared from cells and processed as previously reported. BrieflyUnivariate Cox models were used to analyze the correlation between the IC50 for the drug and the expression of each biomarker in the cell line panel. For each drug, we therefore performed 15 Cox regression analyses, one for each biomarker. The p-values obtained in the Cox regression analysis were used to determine the relevance of the biomarker for predicting the sensitivity to the drug.The experimental research on mice performed in this work complied with institutional, national, and international guidelines for the welfare of animals and was approved by the local ethics committee /CEI HU Virgen Del Rocío/IBIS).7 cells in 0.2 ml of a mixture  of Matrigel basement membrane matrix  and serum-free medium. When the tumors reached approx. 150 mm3, the mice were randomly assigned into treatment or control groups. Zalypsis® was intravenously administered either in 3 consecutive weekly doses (0.9 mg/kg/day) or in 2 cycles of 5 consecutive daily doses (0.3 mg/kg/day). Control animals received an equal volume of vehicle. Caliper measurements of the tumor diameters were performed twice weekly, and the tumor volumes were calculated according to the following formula: (a·b)2/2, where a and b were the longest and shortest diameters, respectively. The animals were humanely euthanized, according to Institutional Animal Care and Use Committee of PharmaMar, Inc.  guidelines, when their tumors reached 3000 mm3 or if significant toxicity  was observed. Differences in tumor volumes between the treated and control groups were evaluated using the unpaired t-test. Statistical significance was defined as p < 0.05. The statistical analyses were performed by LabCat v8.0 SP1 .Four to six week-old athymic nu/nu mice (Harlan Sprague Dawley) were s.c. xenografted into their right flank with approx. 0.5-1 × 10in vitro sensitivity after exposure to Zalypsis, trabectedin, and doxorubicin. The panel included 22 genes involved in tumor progression, cell adhesion, cell cycle control, and cell signaling: Apaf-1, APC, cdk4, c-Kit, cyclin D1, E-cadherin, MDM2, MLH-1, MSH-2, p14ARF, p15INK4b, p16INK4a, p21cip1, p27kip1, p53, p73, p85, PDGFRα, p60src, PTEN, pAKT, and β-catenin. Fourteen genes were analyzed by the presence or absence of mRNA, and the remaining genes were evaluated by the protein level  to trabectedin and doxorubicin were determined for comparison. The ICe Table . Response Table .Similarly, the cell line panel showed a broad range of responses to Zalypsis, with a median IC50 of 502 nM range 0.16 to 4933). It is evident that the panel cell lines can be divided into two groups according to sensitivity to Zalypsis, sensitive lines with an IC50 below 1 nM and resistant lines with an IC50 above 100 nM, establishing a more than 100-fold difference between the sensitive and resistant groups. Although liposarcoma-derived cell lines are highly sensitive to trabectedin and Zalypsis, there is no tissue pattern for the response, and the sensitivity to Zalypsis did not correlate with that for trabectedin (Table 6 to 493350) and the expression pattern of each marker across the cell line panel. Therefore, we performed 15 Cox regression analyses, one for each marker. We then compared the relevance of each molecular marker to the sensitivity to trabectedin or Zalypsis and compared with another 12 drugs previously analyzed[in vitro and in patients previously described and indicating that they might be complementary to doxorubicin in treating solid tumors, especially sarcoma.To explore trabectedin and Zalypsis sensitivity further, we analyzed the relevance of each of the molecular markers for trabectedin and Zalypsis responses. To this end, we performed univariate Cox regression analyses to determine the relationship between sensitivity to Zalypsis  and the expression pattern of each biomarker. As expected due to previous work[Next, we used the 15 independent univariate Cox regression analyses to determine the relationship between sensitivity to trabectedin or Zalypsis  Figure A and B. in vivo validation.Our aim was to identify a predictive biomarker that allows the use of Zalypsis in a personalized manner and, if possible, to broaden the spectrum of tumors that can be treated with this drug. To validate the signature obtained in the sarcoma cell lines, we selected 9 new cell lines of other solid tumor origin, which, different from the low-passaged sarcoma cell lines, can also grow as xenografts and therefore can also be used for in vitro  and the total levels at 10% or 0.5% serum Figure . We foun Figure . HoweverIn vitro studies indicated that high levels of RTK phosphorylation and activation determined resistance to Zalypsis treatment. With the aim of validating this marker in vivo, we generated xenografts from the 9 cell lines that can be xenografted in mice. We used these cells because the low-passage sarcoma cells used in the predictive panel do not grow exponentially when xenografted in immunosuppressed mice. The xenografts of the different cell lines were treated with Zalypsis either at 0.3 mg/kg/day, qdx5x2. or 0.9 mg/kg/day, qdx7x3. A positive response was considered a reduction higher than 60% of the tumor burden with respect to the control, untreated tumor.Of the 9 cell line xenografts tested, 5 responded to treatment with Zalypsis, and 4 did not respond. The non-responding xenografts comprised CALU6, HGC27, SW1990, and SKOV-3 cells  or absence (-) of different mRNAs in our cell line panel. The numbers identify the relative levels of the protein in each case.Click here for file


Cancer cell lines are used extensively to study cancer biology and to test hypotheses in translational research. The relevance of cell lines is dependent on how closely they resemble the tumors being studied. Relating tumors and cell lines, and recognizing their similarities and differences are thus very important for translational research. Rapid advances in genomics have led to the generation of large volumes of genomic and transcriptomic data for a diverse set of primary cancer samples, normal tissue samples and cancer cell lines. Hepatocellular Carcinoma (HCC) is one of the most common tumors worldwide, with high occurrence in Asia and sub-Saharan regions. The current effective treatments of HCC remain limited. In this work, we compared the gene expression measurements of 200 HCC tumor samples from The Cancer Genome Atlas and over 1000 cancer cell lines including 25 HCC cancer cell lines from Cancer Cell Line Encyclopedia. We showed that the HCC tumor samples correlate closely with HCC cell lines in comparison to cell lines derived from other tumor types. We further demonstrated that the most commonly used HCC cell lines resemble HCC tumors, while we identified nearly half of the cell lines that do not resemble primary tumors. Interestingly, a substantial number of genes that are critical for disease development or drug response are either expressed at low levels or absent among highly correlated cell lines; additional attention should be paid to these genes in translational research. Our study will be used to guide the selection of HCC cell lines and pinpoint the specific genes that are differentially expressed in either tumors or cell lines. Moreover, not all cancer cell lines have equal value or relevance as tumor models [Cancer cell lines are widely used to study cancer biology and to test hypotheses in translational research ,2. Numerr models . Therefoet al. recently evaluated ovarian cell lines as tumor models by comparing their genomic profiles published in CCLE and TCGA, and they found that some rarely used cell lines might be more appropriate to study ovarian cancers [It has been shown that genetic and epigenetic changes including gene mutations, deletions, amplifications, translocations and methylation status found in lung tumors are retained in lung cancer cell lines . Prolong cancers . To our in vitro models. However, that these best drug candidates fail to be effective clinically [Liver cancer is the sixth most frequent cancer globally with higher occurrences in Asia and sub-Saharan regions and is the second leading cause of global cancer deaths. The incidence and mortality of liver cancer have increased in the United States and Europe in the past decade. It was projected to become the third leading cause of cancer-related death by 2030 in the USA . While Hinically  indicatein vitro to in vivo and clinical research.In this work, we compared 200 HCC tumor samples from TCGA to over 1000 cancer cell lines from CCLE using their gene expression profiles, and we found that most HCC cell lines are significantly correlated to the primary tumors in comparison to other cancer cell lines. However, from the set of 25 HCC cell lines, nearly half are not significantly correlated to the tumors. We also found that a small subset of the tumors is not significantly correlated to HCC cell lines. We further identified the differentially expressed genes between the tumor samples and cell lines, and we found that a substantial number of differentially expressed genes are located in the extra cellular space. The genes that are over-expressed in the tumor samples are related to immune response, drug metabolism and ABC transporter. This work helps guide us in optimal selection of cell lines and highlights the differentially expressed genes that play an essential role in translating from We compared 200 HCC tumors profiled by RNASeq from TCGA with 1019 cancer cell lines profiled by microarray from CCLE using the top 5000 varying genes (see Methods). There are 28 cancer cell lines derived from liver tissues and 25 of them are related to HCC. Fourteen HCC related cell lines are significantly correlated to the tumors, and 7 non-liver cancer cell lines are correlated  but has no significant anti-tumor effect clinically [in vitro to in vivo.We find that the differentially expressed genes are primarily located in the extracellular space. Moreover, the genes related to immune systems are over expressed in tumors; an observation was previously reported . Interesinically . The difinically -28. Althin vitro or in vivo studies due to limited experimental capability. Choosing appropriate cell lines for experimental testing is thus of critical importance. Unfortunately, crucial information about cell lines  is often missing or ambiguous in cell line databases and relevant publications, which further confounds the selection process. As the genomic and transcriptomic profiles of cancer cell lines and tumors become more publically available, using gene expression to relate cancer cell lines to tumor samples becomes a more useful tool to inform cell line selection and has been applied to other cancers . AlthouOur gene expression analysis of tumors and cell lines demonstrates that HCC cell lines are very tissue specific. They resemble the majority of HCC tumors using top 5000 varying genes. This conclusion still holds if top 1000 varying genes are used (data not shown). The four widely used cell lines  are highly correlated to the tumors. However, nearly half of the cell lines are strikingly not significantly correlated to the tumors. Poor differentiation or genomic damage by chemotherapy drugs may account for the poor correlations of some cell lines.Our analysis also shows that there is substantial variation of individual tumor samples in regards to their correlations with cell lines. Intrinsic heterogeneity among samples may account for the variation. Specifically, we identify 8 out of 200 tumor samples as outliers that have poor correlations with the known HCC cell lines. It may suggest that the drug tested in these cell lines will most likely have a different clinical effect in the outlier patients. It is interesting to further associate these tumors to clinical features so that this subset of tumors can be easily identified clinically and personalized treatments can be designed. However, the current 8 samples are insufficient to draw a robust conclusion. It may be also interesting to associate them to other genetic features.Even though many HCC cell lines have overall high correlations with HCC tumors, a large number of genes are actually absent or lowly expressed in cell lines. A significant number of these genes are involved in disease development. Some genes are related to immune system and pharmacokinetics including drug metabolism and drug transportation. It's known that immune system and pharmacokinetics are related to drug response . InducinIn summary, by leveraging publicly available transcriptomic data, we relate individual HCC tumors and cancer cell lines. We demonstrate that the most commonly used HCC cell lines closely resemble HCC tumors based on their gene expression, and we also identify nearly half of HCC cell lines do not resemble HCC tumors. Even in the highly correlated cell lines, a significant number of genes that are critical for disease development or drug response are absent or expressed at low levels, therefore, additional attention should be paid to these genes in translational research. Our future work includes covering more tumor samples from pubic sources  and more cell lines (the Sanger Cancer Cell Line project ), analyzhttp://www.broadinstitute.org/ccle). Gene symbols in the expression file were converted into GeneIDs using AILUN [http://www.atcc.org), HSRRB (http://cellbank.nibio.go.jp/english/), and KCLB (http://cellbank.snu.ac.kr).We downloaded the cell line gene expression file (CCLE_Expression_Entrez_2012-09-29.gct) and the annotation file (CCLE_sample_info_file_2012-10-18.txt) from the CCLE website (ng AILUN . In totahttp://gdac.broadinstitute.org). The RSEM abundance estimation processed by the TCGA workgroup was used in the following analysis. The sample clinical data was downloaded from TCGA (https://tcga-data.nci.nih.gov/tcga/). In total, we analyzed HCC 200 tumor samples and 50 non-tumor samples. According to TCGA, tumors samples are the primary tumors, namely the tumors at the initial site of cancer. Normal tissue samples are matched to the anatomic site of the tumor but usually not matched to the participant. Without any notes, tumor samples are specifically referred to HCC. We also downloaded all the samples in expO (GSE2109) from Gene Expression Omnibus.The level 3 released gene expressions for RNASeqV2 of HCC were downloaded from GDAC (The top 5000 genes ranked by interquartile range across all cell lines were chosen. Among them, 136 genes that are not matched to any genes in tumor samples were ignored in the following analysis. Since the expressions of tumor samples and cell lines were derived from two different technologies and were transformed via different methods , we usedFor each tumor sample, Mann-Whitney test was used to test the difference of its correlations with HCC cell lines and with all other cell lines. Random 1000 tissue samples from different origins were taken from expO and further used to correct the P value from the Mann-Whitney test. Only the tumors with P value better than 95% of the random tissue samples were considered as being significantly correlated to the HCC cell lines.The common genes between tumor samples and cell lines samples were ranked based on their expression in individual samples. T-test was used to assess the difference of the ranks between tumor samples and cell lines for individual genes and P values were adjusted by the Benjamini-Hochberg for multiple hypothesis testing. Fold change was computed as the ratio between the mean of ranks across tumor samples and the mean of ranks across cell lines. Log2 fold change > 2 and q value <= 0.01 were used to select differentially expressed genes.DESeq  was usedAll the analysis was conducted in R programming language. Bioconductor packages Beanplot, ggplot2 and pheatmap were used for visualization. GO and KEGG pathway enrichment analysis was conducted in DAVID  (BenjamiThe authors declare that they have no competing interests.BC, MS and AJB conceived the study. BC performed the analysis and wrote the manuscript. BC, MS, HFM, DH and AJB discussed the results. All authors contributed to writing, reviewing, editing and approving the manuscript.Lists of differentially expressed genes. Table S1: genes up regulated in HCC tumors comparing to HCC cell lines. Table S2: genes down regulated in HCC tumors compared to HCC cell lines. Table S3: genes differentially expressed in tumors compared to non-tumors. Table S4: genes up regulated in tumors compared to cell lines and dysregulated in tumors compared to non-tumors. Table S5: genes down regulated in tumors compared to cell lines and dysregulated in tumors compared to non-tumors.Click here for file


Pet dogs very frequently develop spontaneous mammary tumors and have been suggested as a good model organism for breast cancer research. In order to obtain an insight into underlying signaling mechanisms during canine mammary tumorigenesis, in this study we assessed the incidence and the mechanism of canonical Wnt activation in a panel of 12 canine mammary tumor cell lines. We show that a subset of canine mammary cell lines exhibit a moderate canonical Wnt activity that is dependent on Wnt ligands, similar to what has been described in human breast cancer cell lines. In addition, three of the tested canine mammary cell lines have a high canonical Wnt activity that is not responsive to inhibitors of Wnt ligand secretion. Tumor cell lines with highly active canonical Wnt signaling often carry mutations in key members of the Wnt signaling cascade. These cell lines, however, carry no mutations in the coding regions of intracellular Wnt pathway components  and have a functional β-catenin destruction complex. Interestingly, however, the cell lines with high canonical Wnt activity specifically overexpress LEF1 mRNA and the knock-down of LEF1 significantly inhibits TCF-reporter activity. In addition, LEF1 is overexpressed in a subset of canine mammary carcinomas, implicating LEF1 in ligand-independent activation of canonical Wnt signaling in canine mammary tumors. We conclude that canonical Wnt activation may be a frequent event in canine mammary tumors both through Wnt ligand-dependent and novel ligand–independent mechanisms. The Wnt signaling is one of the key players during normal mammary gland development as well as during mammary tumorigenesis Many studies have documented active Wnt signaling in mammary tissue based on presence of stabilized β-catenin protein and often in combination with aberrant expression of target genes (e.g. Axin2). Based on these criteria, over 50% of human breast tumor tissue samples assessed, showed signs of aberrant canonical Wnt activity  or its metastasis , benign mixed tumor (CMT-U229) and osteosarcoma-like tumor (CMT-U335) Transfection was performed in FBS-free medium using 3 µl Lipofectamine 2000 (Invitrogen), 800 ng pTOPFLASH (TOP) or pFOPFLASH (FOP)  and 0.5 ng human β-actin-promoter renilla construct http://www.dharmacon.com/designcenter/designcenterpage.aspx . Universal MOCKsiRNA  was used as the negative control for siRNA experiments. There was no cross-silencing of non-target genes checked by blasting the siRNA designed sequences against the canine genome database. The sequence of the LEF1 siRNA duplex is as follows: sense GAAGAGGAAGAGAGAGAAAUU and antisense UUUCUCUCUCUUCCUCUUCUU, and for β-catenin sense GAACGAAGGUGUAGCAACAUU and antisense UGUUGCUACACCUUCGUUCUU. Cell transfections were first optimized with siGLO  (data not shown). 80.000 CMT-U27 cells were transfected with 1 µl DharmaFECT Duo as transfection reagent , 50 nM siRNA and 0.5 µg DNA (TOP or FOP) in 24 wells plates . After 24 h and 48 h incubation in DMEM:F12 and 10% FCS cells were harvested for RNA isolation or TCF-reporter assay.Canine sequence-specific LEF1  and β-catenin  siRNA was designed on the website http://www.gene-quantification.de/rest-mcs.html). For the sequence reactions we used a standard amplification with Phusion-Hot Start Taq  according to manufacturer's protocol. DNA sequence reactions were performed using BigDye v3.1 according to the manufacturer's protocol . All amplifications were performed on an ABI 3130XL  and analyzed in Lasergene (version 9.1 DNASTAR). The obtained sequences were compared with DNA sequences in databases using BLASTn (http://blast.ncbi.nlm.nih.gov/Blast.cgi).From all cell lines, total RNA was isolated from two different passages. From canine mammary tissue and from the mammary cell lines total RNA was isolated and treated with deoxyribonuclease using the RNeasy mini kit  according to the manufacturer's protocol. Quality of mRNA from tissue samples were assessed using a 2100 Bioanalyzer  and RNA integrity number (RIN) of each sample is presented in For whole cell lysis cells were washed with cold HANK's balanced salt solution and scraped in cold RIPA buffer ; 1% sodium dodecyl sulfate (SDS) (vol/vol), 1% Igepal (vol/vol), 0.5% Na-deoxycholate (wt/vol), 1 mM phenylmethylsulfonylfluoride,1 mM Na-orthovanadate and 1 µg/ml aprotinin). After 20 min incubation on ice, samples were centrifuged for 15 min at 16,000 g at 4°C. Protein concentration was determined using Bio-Rad Dc Protein Assay (Bio-Rad Laboratories). Fifty microgram protein of total cell lysates was subjected to SDS-PAGE and analyzed by Western blot. For extraction of cytoplasmic and nuclear protein fractions NE-PER Reagent kit  was used according to the manufacturer's protocol. Ten µg protein was subsequently subjected to western blot analysis. Primary antibodies used in this study were directed against APC (AB-1) Mouse (FE9) (OP44 1∶1000), , β-catenin (Ab6302 1∶4000), human E-cadherin , GAPDH (ab9485 1∶2000) (Abcam) and β-actin pan Ab-5 (MS-1295-P1 1∶2000)(Thermo Scientific) as a reference protein. And as secondary antibody goat anti-mouse HRP-conjugated  was used. HRP was visualized using Advance TM_Enhancedchemiluminescence  and analyzed using GelDoc2000 (BioRad).To quantitatively assess canonical Wnt activity we tested a panel of 12 canine mammary cell lines using a TCF-reporter assay . Three cIWP-2 is a small molecule inhibitor that impairs Wnt ligand palmitoylation and secretion The majority of the canine mammary cell lines used in this study had spindle-cell-like morphology, except for CMT1, CMT-U27 and CMT9, which grow as attached cells but also as partially rounded cells . A partiIn multiple tumors, elevated canonical Wnt activity has been shown to result from mutations in components of β-catenin destruction complex Upon stabilization, β-catenin translocates to the nucleus and interacts with members of TCF/LEF family of transcription factors . Interestingly, although all TCF/LEF family members were expressed in canine mammary cell lines, LEF1 showed high mRNA expression specifically in CMT1, CMT-U27 and CMT9 . DiffereAltogether, this study provides evidence for moderate, ligand-dependent canonical Wnt activation in canine mammary tumors that is comparable to human breast cancer. In addition, we report a novel ligand-independent mechanism involving LEF1 overexpression, which results in high canonical Wnt activity. Our further studies aim to explore this ligand-independent mechanism extensively and to identify the underlying gene mutations.Figure S1Axin2 mRNA expression. Rps19 normalized Axin2 mRNA expression in two different passages of canine mammary cell lines. Cell lines were divided in three groups (from left to right): cell lines with high, moderate or absent canonical Wnt activity.(TIF)Click here for additional data file.Figure S2Expression of TCF-family members upon LEF1 knock-down. Relative Rps19 normalized mRNA expression of LEF1, TCF1, TCF3 and TCF4 24 h after LEF1 knock-down in CMT-U27 cells. Average expression of control conditions for each target gene is set to 100.(TIF)Click here for additional data file.Table S1Sequencing results of target gene coding regions in canine mammary tumor cell lines.(DOCX)Click here for additional data file.