Objective: Deregulation of the cell cycle and apoptosis mechanisms in normal cells causes many problems, including cancer. In this study, a genome-wide expression analysis of cell cycle- and apoptosis-related genes in corticosteroid-, vincristine-, and melphalan-resistant U-266 multiple myeloma cell lines was conducted. Materials and Methods: Resistant U-266 sublines were induced by application of each drug by stepwise dose increments. Resistance gained by the cells was confirmed with XTT cytotoxicity assay and microarray analyses were carried out. Among the cell cycle- and apoptosis-related gene expressions, alterations of more than 2-fold were considered significant. Results: Cyclin E2 was drastically overexpressed in the vincristine-resistant subline and a general upregulation was observed for various cyclin-dependent kinases. Some of the cyclin-dependent kinase inhibitor encoding genes were downregulated in resistant sublines in general. Tumor necrosis factor receptor genes were generally downregulated in corticosteroid- and melphalan-resistant U-266 sublines. Different types of effector caspases were downregulated in all resistant sublines. Ceramide metabolism genes seemed to be changed in favor of survival, especially in the melphalan-resistant subline. Conclusion: This report shows that different types of chemotherapeutic drugs alter different apoptotic and cell cycle-related gene expressions and, as a result, may cause drug-resistant phenotypes in U-266 multiple myeloma cell lines. Among those gene expressions, the most drastic increase in cyclin E2 could be important for the survival of vincristine-resistant U-266 cell lines, whereas alteration of ceramide metabolism genes could be important in melphalan resistance.  Amaç: Normal hücrelerde hücre döngüsü ve apoptoz mekanizmalarının düzensiz çalışması kanser de dahil olmak üzere pek çok soruna neden olmaktadır. Bu çalışmada; kortikosteroid, vinkristin ve melfalan’a dirençli U-266 multipl miyelom hücre hatlarında hücre döngüsü ve apoptoz ile ilgili genlerin ifade düzeylerindeki farklılıklar incelenmiştir.Gereç ve Yöntemler: İlaç dirençli U-266 hücre hatları her bir ilacın artan dozlarda U-266 hücrelerine uygulanması ile geliştirilmiştir. Dirençlilik gelişimi XTT sitotoksisite testleri ile gösterilmiş ve mikroarray analizi gerçekleştirilmiştir. Hücre döngüsü ve apoptoz ile ilgili olan gen ifadelerinden iki katın üzerinde olan değişiklikler anlamlı olarak kabul edilmiştir. Bulgular: Vinkristin dirençli U-266 hücre hattında siklin E2 gen ifadesinin büyük ölçüde arttığı ve çeşitli siklin bağımlı kinaz genlerinin ifadelerinde genel olarak artış olduğu gözlenmiştir. Dirençli hatlarda, bazı siklin bağımlı kinaz inhibitörü kodlayan gen ifadelerinde azalma saptanmıştır. Tümör nekroz faktörü reseptör genlerinin ifadeleri kortikosteroid ve melfalan dirençli U-266 hücre hatlarında genellikle azalmıştır. Tüm dirençli hücrelerde farklı tiplerdeki efektör kaspaz gen ifadelerinde azalma gözlenmiştir. Seramid metabolizması gen ifadelerinde ise melfalan dirençli U-266 hücrelerinin hayatta kalmalarını sağlayacak şekilde değişimler saptanmıştır. Sonuç: Bu sonuçlar, farklı kemoterapötik ilaçların farklı apoptoz ve hücre döngüsü ile ilgili gen ifadelerini değiştirerek U-266 multipl myeloma hücre hatlarında dirençliliğe neden olabileceğini göstermektedir. Bu gen ifadeleri arasında, siklin E2’deki yüksek artış vinkristine dirençli U-266 hücrelerinin hayatta kalımı için önemli olabilecekken, seramid metabolizması ile ilgili gen ifade değişiklikleri melfalan direnci açısından önemli olabileceği düşünülmektedir.The emergence of drug resistance in tumor cells is a major complication for successful anticancer chemotherapy . The balThe cell cycle governs the fate of the cell . ProgresCell cycle-mediated drug resistance is an important problem that must be overcome in cancer chemotherapy. It is best described as a relative insensitivity to a chemotherapeutic agent because of the position of the cells in the cell cycle. It was demonstrated when flavopiridol exposure was followed by paclitaxel in human gastric and breast cancer cells. The multiple cell cycle effects of flavopiridol, including the inhibition of different CDK activities at the G1 and G2 phases, create cell cycle arrest, which prevents cells from entering the M phase ,9.The process of apoptosis is controlled by a diverse range of cell signals, either extracellularly  or intracellularly  ,11. TherIn addition to changes in the expression levels of particular proteins that are related to cell cycle and apoptosis, multidrug-resistant (MDR) cells exhibit major alterations in their sphingolipid composition. Sphingolipids, which include ceramides and sphingosine, are essential structural components of cell membranes that also have messenger functions that regulate the proliferation, survival, and death of cells ,16. CeraA 2- to 3-fold overexpression of glucosylceramide appears to be a rather general aspect of P-glycoprotein-expressing MDR cells. An increased turnover of ceramide to glucosylceramide may allow MDR cells to escape apoptosis, since ceramide plays a major role in the regulation of apoptosis . GlucosyThis study demonstrates a genome-wide expression analysis of cell cycle, apoptosis, and ceramide metabolism genes in corticosteroid (U-266/Pred)-, vincristine (U-266/Vinc)-, and melphalan (U-266/Melp)-resistant multiple myeloma cells.Cell LinesThe human U-266 multiple myeloma cell line was obtained from Gülhane Military Medical School, Ankara, Turkey. The cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 1% L-glutamine, and 1% gentamicin  and were maintained at 37 °C in a humidified air atmosphere with 5% CO2. Corticosteroid (methyl prednisolone) (U-266/Pred)-, vincristine (U-266/Vinc)-, and melphalan (U-266/Melp)-resistant sublines were developed from the original U-266 cells by applying each drug separately in dose increments. The XTT cell viability assay was performed for original and drug-resistant sublines with the Cell Proliferation Assay Kit . The fold of resistance values of the sublines were previously reported .RNA Isolation, cDNA Synthesis, and Target PreparationRNA isolation from all cells was performed using TRI reagent  according to the manufacturer’s instructions. All RNA samples were prepared as duplicates for statistical analysis. RNA concentrations were adjusted to at least 2.5 µg/µL. cRNA synthesis, target hybridization, and scanning were performed at the Ankara University Biotechnology Institute . cDNAs were synthesized from total RNA by One-Cycle Target Labeling Assay  according to the manufacturer’s instructions. Second-strand cDNA synthesis, biotin-labeled cRNA synthesis, and cRNA fragmentation were performed using the Affymetrix GeneChip Kit. Hybridization was conducted at 45 °C and 60 rpm for 16 h in an Affymetrix Gene Chip Hybridization Oven 640 and the arrays were stained using a hybridization stain kit according to the instructions in the technical manual. Washing and staining were performed in an Affymetrix GeneChip Fluidics Station 450 with EukGE-WS2v5 fluidics script. The arrays were scanned in an Affymetrix GeneChip Scanner 3000.Data AnalysisAffymetrix GeneChip Operating Software and GeneSpring GX 7.3.1 Software  were used for expression data analysis. The data were normalized by robust multichip analysis method. The gene expression levels were calculated at the level of oligonucleotide sets by using the median polish method  and log Alterations in cell cycle- and apoptosis-related genes in drug-resistant U-266 multiple myeloma sublines are shown in Table 1. Different types of cell cycle-encoding genes and CDKs, especially CDK6 (-119-fold) and cyclin D3 (-104-fold), were downregulated, whereas CDK inhibitor encoding genes (CDKN2B and CDKN2A) were upregulated in the corticosteroid-resistant U-266 cell line. However, cyclin E2 and the 2 recently identified subunits of E2F, E2F7 and E2F8, were drastically overexpressed in vincristine resistance. In melphalan resistance, some of the CDK inhibitor encoding genes (CDKN1A and CDKN1C) were downregulated.TNF alpha-induced protein 3 (TNFAIP3) and TNF receptor superfamily member 10d (TNFRSF10) were downregulated in the U-266/Pred subline. However, TNF-inducible gene 6 protein (TNFAIP6) and TNF alpha-induced protein 8 (TNFAIP8) were upregulated in vincristine resistance.Caspase-3, -6, and -7 are called effector caspases, which are downstream caspases that in turn cleave other protein substrates within the cell to trigger the apoptotic processes. A general downregulation of different types of caspases was observed in drug-resistant sublines .The baculoviral IAP repeat-containing (BIRC) group of genes, which belongs to a family that inhibits apoptosis, was overexpressed in corticosteroid and vincristine resistance but not in the melphalan-resistant subline. The Bcl-related genes (BNIP1 and BNIP3) were upregulated in the U-266/Vinc subline whereas they were downregulated in U-266/Pred. The most common Bcl-2 and Bcl-XL genes of this family were not significantly altered in these sublines, as confirmed by RT-PCR and western blot analysis (data not shown). The programmed cell death-related genes (PDCD11 and PDCD4) were also upregulated in the vincristine-resistant subline and downregulated in the corticosteroid-resistant one, which is parallel to Bcl-related gene expressions.Table 1 also shows the altered gene expressions in ceramide metabolism. The LAG1 homolog ceramide synthase 6 (LASS 6), sphingomyelin phosphodiesterase (SMPD3), and degenerative spermatocyte homolog 1 lipid desaturase (DEGS1) genes were upregulated while UDP-glucose ceramide glycosyltransferase (UGCG) and N-acylsphingosine amidohydrolase (ASAH1) genes were downregulated in the corticosteroid-resistant subline; all of these regulations cause accumulation of ceramide inside the cells. However, the LASS 6 and SMPD1 genes were downregulated in melphalan resistance, leading to degradation to ceramide inside the cells. The cyclin E2 gene was 487-fold overexpressed in the U-266/Vinc subline. Cyclin E is a member of the cyclin family, which is required for the transition from the G1 to the S phase . On the TNF acts via the TNF receptor (TNF-R) and is part of the extrinsic pathway for triggering apoptosis . TNFRSF1Different types of the effector caspases that trigger the apoptotic processes were downregulated in all resistant U-266 sublines. BIRC-related genes were overexpressed in corticosteroid- and vincristine-resistant sublines. The BIRC group of genes inhibits apoptosis by binding to TNF receptor-associated factors TRAF-1 and TRAF-2 . BAG andCeramides are a family of lipid molecules. The most well-known functions of ceramides as cellular signals include regulating the differentiation, proliferation, programmed cell death, and apoptosis of cells . IncreasIn conclusion, in the vincristine-resistant U-266 multiple myeloma cell line, cyclin E2 gene expression was drastically increased, whereas ceramide metabolism genes were altered only in melphalan resistance in favor of survival in the U-266 cell line. However, TNF receptor genes were generally downregulated in corticosteroid- and melphalan-resistant U-266 sublines. This shows that different types of chemotherapeutic drugs alter different apoptotic and cell cycle-related gene expressions. On the other hand, all of the anticancer agents studied here are currently used in the clinical treatment of multiple myeloma. In vitro, different drug-resistant U-266 multiple myeloma cell lines show altered patterns of gene expressions related to apoptosis and the cell cycle. From a clinical perspective, this in vitro study may be a guide to clinicians for the development of new treatment strategies in drug-resistant cases. In the clinical setting, patients give different responses to the same antimyeloma regimens. By figuring out the patient profile, like in the case of our in vitro study, different types of drug combinations can be used in order to overcome the drug resistance phenotype. However, the results of this study only provide preliminary insight into this phenomenon.Acknowledgments We gratefully acknowledge the Ankara University Biotechnology Research Center for technical assistance. This study was supported by TÜBİTAK (SBAG 3297), Turkey.Conflict of Interest StatementThe authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.


In Snail1 positive NSCLC lines, the drug combination complementarily enhanced mesenchymal-epithelial transition (MET), increasing both E-cadherin and Plakoglobin expression, and reducing Snail1, FAK and PXN expression. In addition, the drug combination abrogated cell migration and matrigel invasion. The co-inhibition of MAPK and SRC induced strong G1/G0 cell cycle arrest in the NSCLC lines, inhibited anchorage independent growth and delayed tumor growth in H460 and H358 mouse xenografts. These data provide rationale for further investigating the combination of MAPK and SRC pathway inhibitors in advanced stage NSCLC.Oncogenesis in non-small cell lung cancer (NSCLC) is regulated by a complex signal transduction network. Single-agent targeted therapy fails frequently due to treatment insensitivity and acquired resistance. In this study, we demonstrate that co-inhibition of the MAPK and SRC pathways using a PD0325901 and Saracatinib kinase inhibitor combination can abrogate tumor growth in NSCLC. PD0325901/Saracatinib at 0.25:1 combination was screened against a panel of 28 NSCLC cell lines and 68% of cell lines were found to be sensitive (IC Lung cancer is one of the leading causes of cancer mortality in many countries –3. ApproThe introduction of targeted therapeutics for cancer treatment in recent years has significantly changed the practice of medical oncology and many targeted therapeutics are being validated in various stages of clinical development. Some of these targeted compounds, such as Gefitinib and Erlotinib, have been approved for first-line treatment to treat advanced NSCLC harboring sensitive epidermal growth factor receptor (EGFR) mutations , 6. UnfoStudies have therefore begun to investigate on the application of targeted drug combinations to abrogate signaling pathways cross-talk and restore treatment sensitivity , 11. Forin situ carcinoma cells to dissociate and become motile, leading to localized invasion and metastatic spread. Indeed, bone, brain, lymph nodes, liver and adrenal glands metastases are a very common secondary localization of disease in lung cancer patients, with 30–40% of patients developing brain and bone metastases in the course of their disease [Studies have also focused on non-genetic mechanisms such as Epithelial Mesenchymal Transition (EMT) as a cause of tumor cell dissemination and drug resistance in cancer therapy , 12–15.  disease , 17. Tar disease , 15. We  disease .In this study, we investigate whether PD0325901 and Saracatinib co-treatment can synergistically suppress cell proliferation and tumorigenicity in NSCLC lines. We also evaluate the impact of PD0325901 and Saracatinib in modulating the EMT process via induction of Mesenchymal-Epithelial Transition (MET) in NSCLC lines. Specifically, we also determine whether PD0325901 and Saracatinib in combination can induce strong antitumor and MET response across multiple NSCLC lines.50 < 2 μM), while 15 cell lines (54%) were considered resistant to this compound (cell proliferation IC50 > 10 μM) , to cell lines that were highly resistant . For Saracatinib single drug treatment, 9 cells lines (32%) were observed to be sensitive, while 11 cell lines (39%) were found to be resistant  to 33 μM (H460). No correlation between the cell lines’ sensitivity to these two compounds was observed.We investigated on the proliferation inhibition effects of PD0325901 and Saracatinib as single drug therapies on a collection of 28 lung cancer cell lines. We found that only 8 out of 28 cell lines (29%) were sensitive to PD0325901 treatment (cell proliferation ICμM) Fig. . In geneant Fig. . The groWe next investigated on the proliferation inhibition effects of PD0325901 (PD) and Saracatinib (AZ) co-treatment on the lung cancer cell lines. We generated the drug response profiles of three different PD0325901 / Saracatinib co-treatments at fixed PD:AZ combination ratios of 4:1, 1:1 and 0.25:1 for each cell line Fig. . We thenin vitro combination studies on these cell lines will be tested at the PD:AZ optimal drug ratio of 0.25:1, as observed previously.To further our investigation on whether PD0325901, Saracatinib and their combination treatment could induce MET, we selected 6 cells lines  which exhibited positive basal EMT transcription factor Snail1 expression. In addition, PD0325901 and Saracatinib combination treatment had presented good synergism on these cell lines Fig. . SubsequWe first demonstrated that MEK inhibition by PD0325901 at 1 μM can potently nullify the phosphorylation of its immediate downstream target ERK1/2 in these cell lines  assays and Boyden chamber Matrigel invasion assays on these cell lines to determine whether the MET induction by PD0325901 and Saracatinib could promote a functional response by limiting the cell migratory and invasive behavior. Both PD0325901 and Saracatinib induced MET were able to significantly reduce the cell migratory behavior of the cell lines compared with untreated controls Fig. ; Fig. 4BFor invasion assay, Matrigel-invasive cell lines Calu-1, H23 and H838 were tested Fig. ; Fig. 4DWe next investigated on how cell cycle progression was regulated by PD0325901 and Saracatinib in these NSCLC lines. Cell-cycle analysis from Propidium Iodide staining experiments indicated that both PD0325901 and Saracatinib exerted predominantly cytostatic effect and induced significant G1-phase accumulation in the cell lines Fig. ; Fig. 5B50 < 1 μM. PD0325901 treatment alone, on the other hand, was potent in reducing anchorage-independent colony growth, with all cell lines responding with IC50 < 1 μM  that were able to grow under anchorage-independent conditions in soft agar assays , we obse0 < 1 μM . This co0 < 1 μM . The PD0in vivo in H460 and H358 xenograft models, using doses previously documented to result in therapeutically-relevant concentrations [We evaluated the single and combinatorial effects of PD0325901 and Saracatinib co-treatment trations , 22. TumWe observed through immunohistochemistry staining that E-cadherin expression was significantly induced in the H460 tumors treated with PD0325901 or Saracatinib, compared with vehicle control. Interestingly, the PD0325901 / Saracatinib combination treatment also induced a moderately higher induction of E-cadherin, compared with the single drug treatments Fig. ; Fig. 6EThe present study shows that SRC and MEK co-inhibition by Saracatinib and PD0325901 respectively can be broadly effective in tumor growth control of a wide panel of NSCLC cell lines. In addition, we show that PD0325901 and Saracatinib are potent EMT modulators and both are effective in inducing a MET response in Snail1 positive NSCLC cell lines. Furthermore, we demonstrate that the co-inhibition of SRC and MEK pathways results in a MET phenotype that is accompanied by a decrease in Snail1 and an increase in E-cadherin expression with low cell migratory ability. These data provide proof-of-concept for the use of PD0325901 and Saracatinib in combination to combat invasive growth in NSCLC.in vivo experiments also confirmed that Saracatinib treatment alone only resulted in modest growth delay with no tumor shrinkage  repository. The PC-9 cell line was a gift from A. Ali . Cell lines were authenticated by DNA short tandem repeats analysis using GenePrint 10 kit (Promega). The cell lines were maintained in RPMI-1640  growth medium supplemented with 10% fetal bovine serum , 2 mM L-glutamine  and 100 U/ml penicillin-streptomycin  at 37°C in a 5% COSaracatinib (AZ) and PD0325901 (PD) were purchased from Selleck Chemicals. Cells were plated into microtiter culture plates (Greiner) and treated with varying concentrations of PD or AZ alone, or drug combinations at fixed PD to AZ ratios. After 72 h, cells were lysed with CellTiter-Glo luminescent cell viability assay reagent (Promega) and luminescence was read using a microplate reader . Percent cell proliferation was calculated relative to DMSO control treated cells.50 to IC80) for each treatment condition were then calculated and tabulated for synergism analysis.Sigmoidal dose-response curve fitting for each treatment condition were analyzed using GraphPad Prism 5 software. Drug concentrations that result in cell proliferation inhibition of between 50% and 80% , was tabulated for the drug combination treatment on each cell line.Combination index (CI) for PD and AZ combination was calculated based on the Loewe Additivity equation . BrieflyE-cadherin antibody was purchased from BD Biosciences. Plakoglobin and Paxillin (PXN) antibodies were purchased from Millipore. Antibodies against Snail1, mitogen-activated protein kinase (ERK1/2), phospho-p44/42 ERK1/2, SRC, phospho-SRC (pY416), focal adhesion kinase (FAK), phospho-PXN (pY118), phospho-Akt (pS473), β-actin and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology. Phospho-FAK (pY861) antibody was purchased from Thermo Scientific.Cells are grown on 6-well plates and treated with compounds or DMSO as control for 48 h. Cells were rinsed with PBS and lysed with RIPA buffer supplemented with protease / phosphatase inhibitor cocktail (Promega). Protein concentration was quantified and equalized protein loads were resolved with Bio-Rad SDS-PAGE system using 8% to 12% polyacrylamide gels and transferred to PVDF membranes. Immunoblotting were performed with the antibodies listed above and bound antibodies were detected by chemiluminescence using Amersham ECL Prime Western Blotting reagent .Cells were plated into black, clear bottom 96-well microtiter plates (Greiner). When the cells reach about 50% confluency, the cultures were then treated with compounds or DMSO as control. After 48 h treatment, cells were gently rinsed with PBS, fixed with 4% paraformaldehyde in PBS for 15 min and permeabilized with ice-cold methanol for 10 min. Samples were then blocked with blocking buffer  for 30 min, followed by immunostaining with E-cadherin (1:500) and Plakoglobin (1:500) primary antibodies in blocking buffer overnight at 4°C. Samples were then incubated with Alexa Fluor 488 secondary antibodies  for 2 h at 25°C, followed by Hoechst 33342 nuclear staining  for 15 min. The wells were then washed and kept in PBS. The plates were sealed with aluminum sealing films  and stored at 4°C prior to fluorescence imaging.Plates were imaged using a confocal microplate imager  with 40× objective, 405 nm laser / 417–477 nm filter cube configuration for nuclei imaging, and 488 nm laser / 525/50 nm filter cube configuration for E-cadherin and Plakoglobin imaging. At least 8 sites were imaged for each well. Image sets were analyzed with ImageJ software using an image processing workflow described previously , 43. ObjT1 − AreaT2) / AreaT1 × 100%.Cells were plated into 6-well plates and grown to confluency. A 200 μl pipette tip was used to make linear scratches on the cell monolayer. The wells were then gently washed with media to remove unattached cells and refreshed with culture media containing test compounds or DMSO control. Suitable areas centered on scratch regions with appropriately 0.60 – 0.75 mm wound gap, were selected for wound closure observation. Images of selected areas were acquired at 0 h (T1) and at the end of the cell migration experiment , using an optical imaging microscope at 4× magnification (Olympus CKX41/C-5060). The wound gap area for each image was measured using ImageJ software. For each T1 and T2 image set, the wound closure percentage was calculated using the formula: . The number of cells in each image was then counted using ImageJ software. Percent cell invasion was calculated relative to DMSO control treated cells.Cell invasion assay was performed using 8 μm pore, 24-well format transwell inserts (Corning). Transwell membranes were coated with 0.1 ml of 0.25 mg/ml matrigel (BD Biosciences) in PBS and incubated overnight at 37°C. The following day, 40 × 10Cell cycle fractions were determined through propidium iodide (PI) nuclear staining. Briefly, cells are grown on 6-well plates and treated with compounds or DMSO as control for 48 h. Cells were then harvested, fixed with 70% cold ethanol, and stained with PI staining buffer  for 30 min at 25°C. Flow cytometry was performed using BD LSR II flow cytometer (BD Biosciences). At least 10,000 cell events were collected per sample. Cell cycle analysis was performed using FlowJo software (Tree Star).2 incubator for a period of 2 – 4 weeks to allow cell colonies to grow large enough to be visualized through MTT tetrazolium dye staining. At the assay endpoint, 0.04 ml of 5 mg/ml MTT solution (Sigma) were added into each well and further incubated at 37°C for 3 h. Cell colony images of each well were acquired using a flatbed scanner (Epson). The number of colonies formed in each well was then quantified using ImageJ software. Percent cell colony formation was calculated relative to DMSO control treated cells.0.5 ml of cell suspension in 0.36% agarose containing culture media was plated into each well on the top of existing 0.6% bottom agarose in 24-well tissue culture plates. 0.5 ml of culture media containing compound or compound combinations at various doses was then loaded into each well. Plates are incubated in a 37°C, 5% CO6 cells/ml, and 0.1 ml cell suspension was injected subcutaneously into the flanks of each animal. The tumors were measured with vernier calipers twice a week and the volume was calculated using the modified ellipsoidal formula (length × width2)/2. Median tumor size at initiation of drug treatment was 50 mm3 (for H460 cells) and 200 mm3 (for H358 cells). Both PD and AZ were prepared in 1% polysorbate 80 (Sigma). The mice were randomized into four groups of five mice and gavage fed with vehicle control, or 12.5 mg/kg PD, or 50 mg/kg AZ, or 12.5 mg/kg PD and 50 mg/kg AZ combination. All drugs were administered once a day, 5 days a week. The mice were euthanized and the xenograft tumors harvested after 3 weeks of drug treatment.All animal work adhered to the Institutional Animal Care and Use Committee (IACUC) guidelines on animal use and handling. Female athymic BALB/c nude mice between 6 to 8 weeks old were maintained and handled in a pathogen-free environment under controlled conditions and received food and water ad libitum. Subconfluent H460 or H358 cells were resuspended in PBS at 40 × 10Xenograft tumors from control and compounds administered mice were fixed in formalin immediately after surgical excision, and subsequently paraffin embedded and sectioned. Tumor sections were deparaffinized using standard histologic procedures and stained with E-cadherin antibodies. Color development was performed using EnVision+ System-HRP (DAB) kit  according the manufacturer's recommendation. Cell nuclei were counterstained with Hematoxylin (Thermo Scientific). Slide images were acquired with an optical imaging microscope at 20× magnification (Olympus IX71/DP71). Images were acquired from 3 tumor samples each for every treatment group.Images were analyzed with ImageJ software using an image processing workflow described previously , 44. BriFor gene knockdown, CDH1 siRNA (sequence: 5′-GGCCUGAAGUGACUCGUAATT-3′) and AllStar negative control siRNA were obtained from Qiagen. siRNA transfection was conducted with JetPRIME reagent (Polyplus Transfection). For gene overexpression, CDH1-GFP and p-CMV-entry empty vector plasmid constructs were obtained from OriGene Technologies. Plasmid transfection was conducted with ViaFect reagent (Promega).t-test for all other results. All statistical tests were 2-sided and the significance level was set at P < 0.05.All data were presented as mean ± standard deviation (SD). The statistical significance of the data obtained was analyzed by two-way ANOVA for tumor xenograft results and Student's


The down-regulated pathway “ribosome” was followed up by measurement of RNA- and protein content. In summary, IPEC-J2 is a morphologically and functionally more differentiated cell line in comparison to IPEC-1. In addition, IPEC-J2 cells are a preferential tool for in vitro studies with the focus on metabolism.The pig shows genetical and physiological resemblance to human, which predestines it as an experimental animal model especially for mucosal physiology. Therefore, the intestinal epithelial cell lines 1 and J2  - spontaneously immortalised cell lines from the porcine intestine - are important tools for studying intestinal function. A microarray (GeneChip Porcine Genome Array) was performed to compare the genome wide gene expression of IPECs. Different significantly up-regulated pathways were identified, like “lysosome”, “pathways in cancer”, “regulation of actin cytoskeleton” and “oxidative phosphorylation” in IPEC-J2 in comparison to IPEC-1. On the other hand, “spliceosome”, “ribosome”, “RNA-degradation” and “tight junction” are significantly down-regulated pathways in IPEC-J2 in comparison to IPEC-1. Examined pathways were followed up by functional analyses. ATP-, oxygen, glucose and lactate-measurement provide evidence for up-regulation of oxidative phosphorylation in IPEC-J2. These cells seem to be more active in their metabolism than IPEC-1 cells due to a significant higher ATP-content as well as a higher O Firmicutes and Bacteroidetes phyla [There is increasing need for suitable enterocytic cell cultures of the jejunum and ileum. Because intestinal disorders are a significant cause of morbidity and mortality in the population world wide, the knowledge of the molecular and biological epithelial cell functions is therefore of special importance. The use of the omnivorous pig as a model to mimic the human intestinal barrier function is given through the anatomical and physiological similarities. The abdominal organs like stomach, spleen, bile duct system, small intestine, kidney and bladder found in pigs are basically the same found in human . Furtheres phyla  colonisein vivo.However, the number of cells available for these cultivation methods is limited. The human intestinal Caco-2 cell line has often been used to study the differentiation of epithelial cells , enzymesIn the recent years the intestinal porcine epithelial cell lines IPEC-1 and IPEC-J2, derived from pig small intestine, have been used especially for epithelial microbiological exposition . Their min vitro model comparable with intestinal physiology.For better understanding of the IPEC-cell lines in terms of growth, metabolism and suitability for microbial and drug testing, in the present study both IPEC-1 and IPEC-J2 were analysed for gene expression , morphology, metabolism as well as cytoskeleton and tight junction structure. Based on microarray analysis, different genes were selected for brush border, structure proteins, cell cycle, metabolism and tight junction proteins, and the protein content in the cells was determined. The final aim of this study was to characterise these cell lines to provide a well-defined 5/cm2 on permeable support . DMEM/HAMs F12 supplemented with 5% foetal calf serum (FCS), 5 ml/500 mL cell culture medium, 16 mM 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid (HEPES)  and 5 ng/mL epidermal growth factor  was used as culture medium. Cells grew at 39°C in an atmosphere of 5% CO2 and 95% relative humidity. The transepithelial electrical resistance (TEER) was measured on day 7, 8, 9 and 10 of culture using a Millicell-TERS-electrode . Prior to the measurement, the electrode was washed in 70% alcohol and ampuwa , then the electrode was washed in pre-warmed medium and the TEER was determined. The same order of alcohol, ampuwa and pre-warmed medium was used to clean the electrode between the measurements. For the analysis of p53 cells were treated with p53 activator  for 24 h.Intestinal porcine epithelial cells  consists of “Feeder agar” (0.5% agar) and “Soft agar” . Both–“Feeder agar” and “Soft agar”–depend on DMEM/HAMs F12 supplemented with 5% FCS, penicillin (100 units/mL) and streptomycin (100 μg/mL). “Feeder agar” and/or “Soft agar” were supplemented with 5 ng/mL EGF and 5 mL/500 mL ITS  to check for possible effects of the supplementation. The “Feeder agar” was poured into a 25 cmTRIzol reagent  was added to samples of IPECs as described in the manufacturer’s protocol. On day 10, cells were scraped from the support and lysed. In the next step, chloroform was added to the cell suspension and RNA recovered from the aqueous phase by precipitation with isopropyl alcohol. The dried pellet was dissolved in diethylpyrocarbonate (DEPC) water . After DNaseI treatment the RNA was cleaned up with the RNeasy Kit . The quantity of RNA was determined using the NanoDrop ND-1000 spectrophotometer  and the integrity was checked by running 1 μg of RNA on a 1% agarose gel. The RNA samples were stored at -80°C until processing.The microarray was performed in three independent experiments. Briefly, Gene Chip Porcine Genome Array  was used, loaded with 500 ng RNA and analysed by provided software (Affymetrix GCOS 1.3) using global scaling to a target signal of 500. The array analyses were performed as described in Diesing et al. (2012) . The michttp://www.bioconductor.org/), employing methods described before [Quality control, data preprocessing and statistical analysis were performed using the R statistical language . Mean values were calculated over all corresponding Affymetrix IDs. A t-test was used to assess statistical significance of differentially expressed genes (p < 0.05) between the cell lines. The resulting lists of up and down-regulated genes was used to identify functional pathways in both cell lines using the DAVID Bioinformatic resources [http://www.genome.jp/kegg/). Our microarray data are deposited under the Gene Expression Omnibus (GEO) with the acc.no. GSE67407 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE67407).In the next step, Affymetrix IDs were mapped to the corresponding gene symbols based on the assignments available from the Ensembl database .The qPCR was performed as described in Nossol et al. (2011) . The QubZonula occludens protein 1, 1:500), mouse anti-human Occludin , rabbit anti-human claudin-3, mouse anti-human claudin-4 , mouse anti-human CK18 , mouse anti-human p53 , mouse anti-human β-actin , rabbit anti-mouse BAD , rabbit anti-human BAX , mouse anti-human BCL-X , mouse anti-human ezrin ; mouse anti-chicken villin ; goat anti-human RPL10a  and rabbit anti-human COX5B . The secondary antibody was purchased with the BM Chemiluminescence Western Blotting Kit mouse/rabbit . Blots were analysed on an Alpha-Ease FC Imaging System .Western blot analyses (n = 3) were performed as described in Nossol et al. (2011) , and theRED (ch 2) and 415–457 nm for DAPI (ch 3)). After scanning images were processed contrast and brightness levels of individual channels using either ImageJ  or Photoshop CS 5 ,IPEC monolayers were fixed and stained as described in Nossol et al. (2011) . BrieflySamples were treated and analysed as described in Nossol et al. (2011) . The semCells were seeded on 12-well-ThinCerts for 10 days. On day 9, cells were treated with Carbonylcyanid-4-trifluormethoxyphenylhydrazon  for 24 h. On day 10, media was removed and a boiling hot puffer  was added to the cells. In the next step, cells were scraped off the membrane with a cell scraper. The cell suspension was transferred into a tube, incubated for 2 min at 100°C, centrifuged at 1000xg for 60 s. Supernatants were pipetted into a white 96-microplate . Samples kept on ice until measurement. An ATP-standard curve was prepared after manufacturer’s instructions .The oxygen uptake was measured with a Microx TX3 . Microx TX3 is a micro fiber optic oxygen meter with a microsensor based on a 140 μm optical fiber. The sensor was calibrated after manufacturer’s instructions . The oxygen content in the medium without cells and the oxygen content in the medium  and ThinCerts). The oxygen uptake is given due to the difference between both values .Lactate and glucose content of both cell lines was analysed on day 10 of cultivation. Therefore, apical and basolateral supernatants were collected of both cell lines. Medium was changed 72 h before and medium without cells was used as blank. All samples were stored on ice and immediately measured with a Roche/Hitachi Cobas c system . The difference between blank and sample represents the glucose consumption respectively lactate production of the cell.Results of the ATP, Glucose and Lactate measurement, protein and RNA-content are expressed as means and standard deviation (S.D.) of at least 5 independent experiments. Asterisks indicate significant differences from IPEC-1 . The analysis of the ATP-content of both cell lines was examined using an univariate analysis of variance .Kyoto encyclopedia of genes and genome) pathways in IPEC-J2 in comparison to IPEC-1 are shown in Both cell lines were cultured on membranes for 10 days. The analysis of the gene expression profile of IPEC-1 and IPEC-J2 aimed to generate a list of genes differentially expressed in both cell lines. 14 746 genes were analysed and 5 787 varied significantly. We observed 2 631 genes up-regulated and 3 156 genes down-regulated in IPEC-J2 in comparison to IPEC-1. Up- and down-regulated KEGG .IPEC-1 and IPEC-J2 form a continuous monolayer characterised by polarised growth, transepithelial resistance (TEER) and the expression of actin, villin-1 and villin-2 (= ezrin) . The polIn the next step, other genes of cytoskeleton proteins that are in close contact to actin and important markers of the brush border were analysed. VIL1 (Villin 1), VIL2 (Villin 2 = Ezrin), TLR4 (Toll like receptor 4), ESPN  and MUC4  in qPCR analyses in comparison to IPEC-1. Here, we found a 27-fold increase in the microarray analyses and a 300-fold increase in the qPCR ; Table 3The pathway “RNA degradation” was significantly down-regulated in IPEC-J2. Furthermore, we examined the RNA-content per cell and compared both cell lines. IPEC-J2 showed a significant higher RNA-content than IPEC-1 p<0.001; . This goA more detailed examination of the morphology of IPEC-1 and IPEC-J2 showed the clear network of the membrane-linked proteins involved in the cell-cell-contacts. 28 genes are regulated in the microarray analyses that are involved in the network of the connected cells. Claudin-1, claudin-3, claudin-7 and occludin were chosen as marker for cell-cell contacts and tight junctions. An increased mRNA-level of claudin-3 and claudin-7 . Similar results were found with the focus on lactate-production . Furthermore, the microarray analyses showed 29 significantly regulated genes like COX5B, COX7A2, COX7C, COX8A, ATP5D, ATP5E, ATPH, NDUFA2, NDUFA4 and NDUFB2. In addition, important genes of the aerobic metabolism like CYC1, PDHB and SDHB were examined , the decoupling of the respiratory chain was performed using Carbonylcyanid-p-trifluoromethoxyphenylhydrazon (FCCP). The results in ATP-production are given in in vitro test of cancer. Normal epithelial cells are supported by a basement membrane, which provides survival and proliferative signals, and they undergo apoptosis when seeded in suspension culture [Only a few non-transformed intestinal cell lines such as IEC-6 and IEC-18 exist . In 1989 culture .Furthermore, the absorptive epithelium of the gastrointestinal tract developed the brush border, a highly specialised apical membrane that increases the surface area. A well-developed brush border is an important marker of polarised intestinal epithelial cells. TEM analyses at different time points showed a two-fold higher number of microvilli in IPEC-J2 than in IPEC-1 at both time points . These microvilli are primarily composed of six proteins: actin, fimbrin, villin, brush border myosin (Myo1A), calmodulin and a non-erythrocytic spectrin . This stMany cancer cell lines like Caco-2 and HT-29 also express high level of villin because of their ability to develop an enterocytic differentiation . We had Zonula occludens (tight junctions), Zonula adhaerens and the basally localised desmosomes are commonly found between epithelial cells in vivo. Furthermore, Zonula adhaerens and Zonula occludens serve as structurally landmark for cell polarisation due to the defining of an apical/basolateral axis [Zonula occludens regulate barrier function [Differences between the cell lines were found in the tight junctions (down-regulated in IPEC-J2). ral axis . Proteinfunction . In this2- and glucose-consumption and lactate-production. IPEC-J2 showed a significantly higher ATP-content than IPEC-1. FCCP results in a significantly decreased ATP-content in IPEC-J2 but not in IPEC-1. These results go along with the O2-utilisation. IPEC-J2 showed a significant higher O2-consumption than IPEC-1 on dishes as well as on membranes. Furthermore, glucose-consumption and lactate-production were examined. A significant higher glucose-consumption and lactate-production were found in IPEC-J2 in comparison to IPEC-1. We suppose that IPEC-J2 used aerobic as well as anaerobic glycolysis to produce ATP due to: 1. high ATP content (aerobic), 2. FCCP results in significantly decreased ATP-levels, 3. high glucose consumption (anaerobic), 4. high O2-consumption (aerobic) and 5. high lactate production (anaerobic). IPEC-1 seems to be in a “steady state” or non-proliferative state because: 1. low levels of ATP, 2. low O2 consumption, 3. low glucose consumption and 4. low lactate production [Furthermore, differences between IPEC-1 and IPEC-J2 were found in two important pathways of cell metabolism namely oxidative phosphorylation and glycolysis. GAPDH, HIF1a and PDH are important genes, which are up-regulated when cells produce ATP via glycolysis. HIF1a is the most important, because it regulates all other genes that are involved in the glycolysis. In our study, we did not detect any differences between the cell lines in HIF-1a-mRNA in the qPCR but an up-regulation in the microarray in IPEC-J2 . In a reoduction . Glucoseoduction , which l2-consumption, glucose-utilisation and lactate production with a significant effect on ATP-production. When designing experiments using epithelial cell lines the present results should be taken into account, especially when analysing the metabolism of the cells, e.g. in terms of consumption of glucose and ATP.In conclusion we demonstrate that both cell lines are differentially regulated in: A. Cytoskeleton with effects on the brush border, B. Tight junctions with no effect on TEER values but possible on ion-transport; C. Metabolism with the focus on O


Gas6 was abundantly present in all specimens. We next tested AXL protein expression immunohistochemically in 36 tumors  from 25 ES patients. Low, medium and high AXL protein expression was observed in 17% (n = 6), 19% (n = 7) and 36% (n = 13) of samples. In primary tumors (n = 15), high AXL expression correlated significantly with a worse overall survival compared to patients with lower expression . No genetic aberrations were detected in the AXL RTK domain (n = 29). The AXL-inhibitor BGB324 affected viability (IC50 0.79–2.13 μmol/L) and migratory potential of all tested ES cell lines in vitro (n = 5–6). BGB324 chemosensitized chemotherapy-resistant ES-4 cells to vincristine and doxorubicin. These data suggest that AXL is a potential novel, druggable therapeutic target in ES.New targets for Ewing sarcoma (ES) patients are urgently needed. Therefore, we investigated the expression and genetic aberrations of the oncogenic receptor tyrosine kinase (RTK) AXL in ES and determined the efficacy of AXL targeting on cell viability and migration. First, AXL and Gas6 (ligand) mRNA expression was determined by RT-PCR on 29 ES samples. Low, medium and high AXL mRNA expression was observed in 31% ( Ewing sarcoma (ES) is a highly aggressive type of primary bone cancer, predominantly affecting children and adolescents. Despite intensive surgery, radiotherapy and polychemotherapy, the final outcome in patients with metastatic disease has not improved impressively during the last decades and side-effects of treatment could be severe , 2. ThisAXL gene was one of many genes found to be significantly overexpressed [AXL (or UFO) is a member of the TAM  RTK family and plays an important role in various cellular processes, including survival, proliferation, migration, invasion and angiogenesis. Growth-arrest-specific protein 6 (Gas6) is the ligand with the highest affinity for AXL. Similar to other RTKs, AXL activation results in intracellular signal transduction involving phosphatidylinositol 3 (PI3)/Akt kinase and extracellular signal regulated kinase (Erk) pathways . Moreovexpressed . In addixpressed .Recently, the first-in class, highly selective AXL tyrosine kinase inhibitor BGB324 (R428) entered phase I clinical studies . Encourain vitro to explore the functional relevance of AXL-targeting in ES.The exact function of AXL in ES is, however, still unclear. We therefore examined AXL and Gas6 mRNA and AXL protein expression levels in a well-defined cohort of human ES specimens and correlated AXL protein expression levels to clinicopathological characteristics and patient outcome. Several tumor samples were examined for genetic AXL aberrations as well. The effect of the AXL inhibitor BGB324 was examined on ES cell viability, chemo-sensitivity and migration n = 14) and 21% (n = 6) of ES samples, respectively. Low expression levels were detected in 31% (n = 9) of cases  and virtually all positive sections demonstrated protein expression in > 70% of tumor cells  and 19% (n = 7) of ES samples, respectively. However, the most outstanding samples were those with high AXL expression, which was observed in 36% (n = 13) of cases. AXL expression levels in these lesions were substantially higher than in samples with medium or lower AXL scores, and therefore seemed to be a distinct group. Also compared to a panel of 80 breast carcinoma (BC) samples stained in parallel, including triple-negative samples and selected positive BC tissue controls, considerably higher AXL expression was observed in ES samples , 11 post-chemotherapy resections, 7 distant metastases and 3 local relapses .In six ES specimens, both AXL mRNA and AXL protein expression levels were evaluated. There was a significant positive correlation between AXL mRNA and protein expression . We could not confirm this finding in a paired analysis, because there were only three patients from which paired primary and post-chemotherapy resection specimens were scored.In the post-chemotherapy resection group, AXL expression was significantly lower compared to expression in the group of primary tumors (Table n = 15) to investigate a possible correlation between AXL expression and patient outcome. No, low, medium and high AXL expression was observed in 7% (n = 1), 20% (n = 3), 20% (n = 3) and 53% (n = 8) of primary tumors, respectively. Because samples with highest AXL expression (score 3) seemed to be a distinct group as described above and to increase statistical power for survival analysis, the primary tumor cohort was divided into two groups: low (scores 0–2) and high (score 3) AXL expression. Interestingly, high AXL expression correlated significantly with a worse OS compared to low expression  (Kaplan-Meier (log-rank test) survival analysis was performed on the primary tumors  Figure . There w = 0.021).Twenty-nine ES patients and six ES cell lines were examined for genetic alterations in mRNA encoding the AXL RTK domain. This domain was selected because it is the intracellular catalytic subunit of the receptor mediating signal transduction, and BGB324 specifically interferes with this domain [in vitro by MTT assays. All cell lines demonstrated AXL protein and mRNA expression, although expression levels varied  and Gas6 stimulation in six ES cell lines d Figure . There wd Figure . Similard Figure . BGB324 3 μmol/L . No corr3 μmol/L .in vitro whether BGB324 synergizes with three commonly used chemotherapeutics in ES, all from different classes of chemotherapy: doxorubicin (anti-tumor antibiotic), vincristine (mitotic inhibitor) and cyclophosphamide  [50 0.58 – 1.72 ng/mL) than to doxorubicin (IC50 5.32 – 59.14 ng/mL) and cyclophosphamide (IC50 225 – 541 ng/mL). The high AXL- and Gas6-expressing ES-4 cell line was the most resistant cell line to vincristine and doxorubicin. The high AXL and low Gas6 expressing EW-8 cell line was relatively the most resistant cell line to cyclophosphamide. The addition of vincristine to BGB324 resulted in synergistic effects (CI < 1) in ES-4 at all but one (nearly additive) tested concentrations, and in the other cell lines at some concentrations  , 25. Thes Figure . In ES-1s Figure . The comIn vitro migration assays were performed with ES-2, ES-4, ES-7, ES-8 and EW-8 cell lines , non-small cell lung cancer (NSCLC), pancreatic cancer and prostate cancer , 26, 27.In this study, AXL was predominantly present in the cytoplasm of ES cells, although membranous, nuclear and vascular expression was occasionally reported as well. This expression pattern closely resembles data in other cancer types –30. Mostin vitro, suggesting ligand-independent signaling of AXL. We tried to assess phosphorylation of AXL (pAXL) in our ES samples and cell lines, but unfortunately technical difficulties precluded these determinations. Evaluation of downstream pathway activation however demonstrated a decrease in pAKT and pERK levels upon BGB324 treatment in some of the cell lines, which is in line with previous data in other cancer types [in vitro, BGB324 did affect ES cell viability and impressively reduced cell migration in the majority of ES cell lines. Accordingly, in osteosarcoma and triple-negative BC cell lines, AXL-signaling pathway interference had considerably greater effects on migration than on proliferation in vitro [In the present study, AXL mRNA expression levels correlated well with protein expression in ES patient samples and cell lines. To further investigate mechanisms underlying aberrant AXL signaling, we screened the AXL RTK domain in several ES patients and in our ES cell lines. However, no aberrations were found, suggesting that ES harbor wild-type AXL, although we cannot exclude aberrations outside the investigated domains. These findings are in line with previous research, since at present no (activating) AXL mutations have been reported in other tumor types as well , 33. Theer types , 32, 35.in vitro , 24.in vivo and in ES patients. In addition to tumor AXL expression, vascular AXL was also reported in ES samples. Numerous preclinical and early clinical data show that targeting of vascular mechanisms could be effective in ES and BGB324 was shown to suppress angiogenesis in in vivo tumor models [50 values in our study are nearly identical to those reported in numerous studies in other cancer types (range 0.81 – 3.15 μM) [in vivo experiments with the same cell lines as tested in vitro and showed effective anti-tumor efficacy of BGB324 at clinically achievable doses [On top of that, there are indications that BGB324 could even be more potent r models . Moreove3.15 μM) , 37, 38.le doses . Furtherin vitro and in vivo results in several tumor types [in vivo ES models that a smaller IGF-1R-targeting compound exerted superior tumor penetrating and thus better IGF-1R-targeting properties than a relatively large IGF-1R-targeting antibody [Although at present no clinical data are available concerning the efficacy and safety of BGB324 in cancer patients, BGB324 was recently evaluated in early clinical safety studies in healthy volunteers with encouraging results. Treatment with BGB324 was established as being safe and well tolerated . Phase Ior types . For ES antibody . In addiAlthough the results of combined BGB324 and chemotherapy treatments were not straightforward by showing different combined effects with different classes of chemotherapy, the combination of BGB324 with vincristine led to the use of less vincristine in most cell lines to obtain a similar response. This could be an important step towards reducing chemotherapy-induced side-effects. Interestingly, in the high AXL- and Gas6-expressing ES-4 cell line, synergistic or additive effects were observed for most tested concentrations of vincristine and doxorubicin. Because ES-4 was the most resistant cell line to these two types of chemotherapy, this suggests that particularly chemotherapy-resistant, thus poor-prognosis, ES patients might benefit from a combination of BGB324 and chemotherapy. Because in our study, apart from the ES-4 cell line, in all other ES cell lines the combination of BGB324 with doxorubicin resulted in antagonistic effects, this also shows that this particular combination should be handled with caution for ES treatment. The latter also applies to the combination of cyclophosphamide to BGB324, since this led to moderate antagonism in all ES cell lines. Although some studies reported potent effects when combining BGB324 to chemotherapy, including additive effects in combination with doxorubicin, a very recent extensive study investigating the effects of different types of chemotherapy (and other drugs) combined to BGB324 in a large panel of human cancer cell lines stress the importance of selecting the appropriate class of inhibitor for combined treatment regimens , 32. SimTo date, IGF-1R is the most intensively studied RTK target in ES. The lack of activity in high volume malignancies and the observed and not fully elucidated primary and acquired resistance to IGF-1R-targeted therapies in ES patients unfortunately led to the suspension of a lot of planned IGF-1R-targeted research. Because AXL is induced during resistance development to IGF-1R-targeted therapies in rhabdomyosarcomas, which may also be the case in ES, this further supports a rationale to target AXL in ES . Furtherin vitro, this may offer great potential. Moreover, because AXL is implicated in chemotherapy resistance, and BGB324 chemosensitized chemotherapy-resistant ES-4 cells to vincristine and doxorubicin, the combination of BGB324 to chemotherapy may be of particular interest to ES patients with a poor response to conventional chemotherapy. Our data also stress the importance of selecting the appropriate chemotherapeutic agent for future combination studies, since different classes of chemotherapy exerted different therapeutic effects in combination with BGB324. Although further (in vivo) research is warranted, we believe that AXL-targeting is a promising line of inquiry for ES treatment and could have a significant impact on future outcomes for the often young ES patients.Altogether, we showed for the first time that AXL is a potential novel and druggable therapeutic target in ES. High AXL expression in primary ES appeared to be a strong independent predictor of poor OS. Since the leading cause of ES patient mortality is metastatic disease and BGB324 clearly inhibited ES cell migration Thirty-six tumor samples from 25 patients diagnosed with ES between 1986 and 2009 were retrieved from the files of the dept. of Pathology of the Radboud University Medical Centre (Radboud UMC), Nijmegen, the Netherlands. In all patients the diagnosis was confirmed by ES typical gene fusions and all were treated at the Radboud UMC. The average follow-up was 66 months (range 2–261 months). The study was performed in accordance with the Code of Conduct of the Federation of Medical Scientific Societies in the Netherlands. Patient characteristics are summarized in Table 2O2 in distilled water for 10 min at room temperature (RT), and nonspecific binding was prevented by blocking with 20% normal goat serum in phosphate-buffered saline-bovine serum albumin 1%  for 30 min at RT. Subsequently, sections were incubated with monoclonal rabbit anti-AXL  antibody in PBS-BSA 1% overnight in a humidified chamber at 4°C. Sections were then incubated with a goat-anti-rabbit biotinylated secondary antibody  for 30 min at RT followed by an incubation with an avidin-biotinylated horseradish peroxidase complex (ABC) using Vectastain ABC kit  for 30 min at RT. Next, the catalyzed reporter deposition technique  was used to enhance the sensitivity of the staining method followed by a second incubation with ABC for 15 min at RT. Finally, antibody binding was visualized by an incubation for 7 min at RT with 3, 3′-diaminobenzidine (Bright-DAB). Slides were counterstained with haematoxylin, dehydrated and coverslipped.In this study we used IHC to specifically investigate AXL protein expression levels and target localization. Selected AXL-positive BC tumor samples and the triple-negative BC cell line MDA-MB-231 served as positive controls and selected AXL-negative BC samples and substitution of the primary antibody by PBS, 1% BSA as negative controls , 47. ConSlides were scored for AXL expression by two independent observers and consensus cytoplasmic scores were given as follows: 0, no positive cells; 1, low; 2, medium; 3, high staining intensity (> 10% of tumor cells).Correlations between categorical data were calculated with Fisher's exact test. Wilcoxon signed-rank test was used to compare paired samples. Linear correlation between two continuous parameters was evaluated using Spearman correlation. Kaplan-Meier and log-rank methods were used to draw and evaluate the significance of survival curves. Multivariate Cox regression analysis was used to adjust for other variables after univariate survival analysis. Survival times are presented as mean survival in months ± SD.AXL RTK domain and Gas6 genes were subsequently amplified by PCR (Reverse transcription polymerase chain reaction (RT-PCR) of the AXL RTK domain and Gas6 was performed on 29 fresh frozen ES tissue samples (> 50% tumor cells) and 6 ES cell lines. Total RNA was extracted with RNA-Bee  according to manufacturer's protocol. Then, first strand cDNA synthesis was performed on 1 μg RNA for 1 h at 42°C with random hexamer primers  and SuperScript II reverse transcriptase . d by PCR . GAPDH wFor Western Blot analysis, protein extracts were purified from ES cell lines. Cells were incubated in ice-cold RIPA buffer containing protease and phosphatase inhibitors and the lysates were centrifuged at 14,000 g at 4°C for 30 min. The protein concentrations of the supernatants were determined with the BCA protein assay system . Of each cell line, equal amounts of protein (50 μg) were loaded and run on a 6% Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) gel under reducing conditions and subsequently transferred to nitrocellulose membranes. After blocking with blockbuffer  in PBS (1:1) at room temperature (RT) for 1 h, membranes were incubated overnight at 4°C with monoclonal rabbit anti-AXL (1:1000), monoclonal rabbit anti-phosphorylated (p) AKT (1:1000), monoclonal rabbit anti-pERK (1:500), polyclonal rabbit anti-pS6RP (1:1000) or monoclonal rabbit anti-p4EBP1 (1:1000). All primary antibodies are from Cell Signaling Technology. Next, the blots were incubated at room temperature for 1 h with a goat-anti-rabbit fluor 680-conjugated secondary antibody , incubated for 1 h at RT with monoclonal mouse anti-α-tubulin  as a loading control and subsequently incubated for 1 h at RT with a goat-anti-mouse fluor 800-conjugated secondary antibody . The fluorescent signals were analyzed with the Odyssey Infrared Imaging System (LI-COR Biosciences) and Odyssey Application Software (version 3.0.30).2/95% air at 37°C.ES cell lines  were generously provided by Peter Houghton of the Pediatric Preclinical Testing Program (PPTP) . The presence of EWS/FLI1 gene fusions was confirmed by RT-PCR in all cell lines in our laboratory prior to the initiation of the experiments. Cells were cultured in RPMI-1640 medium  supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin (Lonza) in a humidified atmosphere of 5% COBGB324 (R428), a well-characterized small molecule inhibitor of AXL, was purchased from Synkinase . BGB324 is unique in its potent on-target activity and restricted selectivity profile [50 values were calculated with GraphPad Prism Version 4.00 software.The effects of BGB324, rhGas6, doxorubicin, vincristine and cyclophosphamide (4-HC) on cell viability were assessed in MTT assays. ES cells were seeded into flat-bottomed 96-well plates at 5,000 cells/100 μl/well and allowed to adhere. Twenty-four hours later, designated drug or ligand doses were added and cells were incubated for 72 h. All drug concentrations and blanks were completed in quadruplicate. Subsequently, 20 μl of 5 mg/ml MTT (Sigma-Aldrich) in PBS was added to each well and cells were incubated for another 3.5 h at 37°C. Afterwards, the medium was carefully removed and the formazan crystals were dissolved in 150 μl of acidified isopropanol solution. Absorbance was read at 560 nm using an ELISA reader. The experiments were repeated in duplo (rhGas6) or triplicate  and ICTo assess drug synergy between BGB324 and doxorubicin, vincristine or cyclophosphamide (4-HC), the combination index (CI) method was used as previously described . In comb6 cells/2 ml/well and allowed to adhere. When 80–100% confluence was reached, two slashes were created in the middle of the wells with the tip of a P-200 pipette. Cells were rinsed with PBS and replaced with 2 ml media containing designated BGB324 doses . After TT assay . The deg


The estrogen receptor-positive M13SV1 breast epithelial cell line was proposed to be a suitable in vitro model for breast cancer research since two derivatives with graduated tumorigenicity—M13SV1-R2-2 and M13SV1-R2-N1—are available for this cell line. In the present study, these three cell lines were comparatively examined for their morphological and their biochemical properties on the molecular level.A transcriptomic approach  was chosen to unravel differences in gene expression among the three cell lines. Network analysis was conducted to identify deregulated signaling pathways. Cellular viability was determined by impedance measurements as well as by neutral red uptake assay. Apoptosis was determined by using a caspase assay. For morphological characterization, cells were grown in three-dimensional cell culture, and cellular differentiation and spheroid formation was followed by immunofluorescence staining by using confocal laser scanning microscopy.The gene array results indicated that there were only marginal differences in gene expression among the three cell lines. Network analysis predicted the R2-N1 derivative (1) to display enhanced apoptosis and (2) to have a higher migration capability compared to its parent cell line M13SV1. Enhanced apoptosis was confirmed by elevated caspase activity, and increased migration was observed in 3D culture when cells migrated out of the globular spheroids. In 3D cell culture, all three cell lines similarly formed spheroids within three days, but there was no acini formation until day 21 which is indicated by a growth arrest around day 15, cellular polarization, and the formation of hollow lumen inside the spheroids. These characteristics, however, are crucial to study, e.g., the differentiation process of breast epithelial cells in vitro.Due to the molecular and morphological features, the M13SV1 cell line and its tumorigenic derivatives seem to be less suitable as in vitro models than other cell lines such as the MCF-10A cell line which displays proper acini formation in 3D culture.The online version of this article (doi:10.1186/s12935-015-0262-5) contains supplementary material, which is available to authorized users. Nowadays, breast cancer research largely depends on the availability of model systems suitable to monitor breast epithelial cell differentiation and crucial characteristics of mammary gland development in vitro. There are two major types of epithelial cells in mammary gland, namely luminal cells and basal or myoepithelial cells. These two cell types originate from the same stem cell population that is also found in the mammary epithelium . For in A transcriptomic approach was chosen to analyze the differences between the M13SV1 cell line and its tumorigenic derivatives on the molecular level. Total RNA was prepared from the three different cell lines and subjected to gene array analysis. By using the standard settings for gene array data evaluation , it turned out that only a small number of the 24,266 different transcripts represented by the gene array were differentially expressed among the three cell lines  was employed to gain insight into molecular metabolic and signaling pathways that are differentially regulated among the three cell lines. The IPA software is a literature-based database that allows a comparison of one’s own experimental data with published data. In our analysis, this comparison was restricted to published experimental data obtained only with human breast cancer cell lines or with primary isolates from human mammary gland. It turned out that although 122 gene transcripts were significantly deregulated in the R2-2 cell line compared to its parent cell line M13SV1  was employed to examine the expression of 84 different breast cancer-related genes in parallel. The results of this analysis are summarized in Table To further characterize the molecular differences between the three cell lines in particular with respect to the increasing tumorigenic potential, qPCR experiments were conducted with a focus on genes related to breast cancer. For this purpose, the Human Breast Cancer & Estrogen Receptor Signaling RTIn regard to the prediction that the highly tumorigenic cell line R2-N1 has an enhanced migration capability compared to the parent cell line, the growth characteristics and the morphology of the three cell lines were compared with each other. Under standard cell culture conditions, there were no apparent differences in cell growth and cell morphology when the cells grew in two dimensions on the bottom of cell culture flasks Fig. a–c. In tConfocal microscopy was applied to further characterize the biochemical and morphological features of M13SV1 cells grown in 3D culture. Antibody staining revealed that stem cell marker such as CD24 and Oct4 were present only at day one after seeding, and that these markers were lost around day 4 in the course of spheroid growth Fig. . Luminalp-dioxin (TCDD) resulted in Akt and ERK2 activation which in turn stimulated cellular proliferation [a)anthracene (DMBA) on the three cell lines were examined, and it was shown that the non-tumorigenic cell line M13SV1 was more susceptible to the mutagen than its tumorigenic derivatives in terms of, e.g., oxidative DNA damage, formation of DNA adducts, and alteration of expression of specific cancer-related genes [The development of well-defined in vitro cell culture models for breast cancer research is a crucial issue not only for fundamental research on mammary gland carcinogenesis but also in terms of, e.g., testing of chemical substances which may have an impact on breast cancer development. With a focus on endocrine disruptors and the examination of potential carcinogenic properties of estrogenic compounds, estrogen-responsive cell lines may be preferred for the development of an in vitro test system. The M13SV1 cell line was regarded as a promising cell culture model as it was shown to be ER positive and even retained some stem cell characteristics. This feature might be regarded as an additional advantage with respect to the stem cell theory which says that numerous tumors originate from stem cells –9. Indeeferation . The autferation . Finallyed genes .The aim of the present study was to compare the M13SV1 cell line with its two tumorigenic derivatives on the molecular level. Thus, the transcriptomes of the three cell lines were compared with each other in order to identify the differences in gene expression which were supposed to be related to the different tumorigenic phenotypes. It has been reported in an earlier transcriptomic study that immortalization of type I HBEC with SV40 DNA to give the M13SV1 cell line was accompanied by the deregulation of 148 genes with a fold change of at least twofold [In order to establish an in vitro test system for human breast epithelial cell lines suitable to monitor substance-induced deregulation of the differentiation process it is crucial to use a cell line which differentiates in 3D culture as similar as possible compared to the in vivo situation in regard to mammary gland formation. Kenny and colleagues have classified 25 breast cell lines into four groups according to their colony morphology . Due to Thus, in conclusion, our analysis revealed that the M13SV1 cell line and its two tumorigenic derivatives seem to be less suitable for the development of an in vitro test system than other permanent HBEC cell lines that belong to the “round” class such as the MCF-12A cell line or the MPE-600 cell line .2.The HBEC lines M13SV1, M13SV1-R2-2 (designated as R2-2 cells in the following) and M13SV1-R2-N1 (designated as R2-N1 cells in the following) were kindly provided by the working group of James E. Trosko . The cells were routinely cultured in phenol red- and hormone-free Michigan State University-1 (MSU-1) medium [For all experiments, cell cultures at a confluence of about 80–90 % were washed once with phosphate buffered saline (PBS) and trypsinized (0.05 % trypsin/0.02 % EDTA). Cells were resuspended in culture medium and trypan blue staining was used to determine viability before seeding. Only cells with a viability of at least 90 % were used for experiments.2). Purification of total RNA was performed using the RNeasy Mini Kit according to manufacturer’s recommendations . At a confluence of about 80–90 % the cells were washed twice with ice-cold PBS, directly disrupted in lysis buffer and homogenized using QIAshredder spin columns . An additional on-column DNAse digestion was performed on all samples to remove residual DNA . The concentration of RNA was determined with a NanoDrop ND-1000 spectrophotometer  and integrity was assessed by using Agilent RNA 6000 Nano LabChip kit with the Agilent 2100 Bioanalyzer according to manufacturer´s protocol . All samples for the microarray analysis as well as PCR array experiments should have a RNA Integrity Number (RIN), a measure of degradation, of 8 or higher .The HBEC lines were seeded in separate cell culture flasks  by using Human Transcriptome 2.0 Arrays (HTA 2.0). 100 ng of total RNA was used for labelling and hybridization according to the protocols of Eurofins. Three independent biological replicates were processed for each cell line. Lists of differentially expressed genes between two cell lines were generated using the Transcriptome Analysis Console (TAC) 2.0 software . The resulting datasets were further processed by using the software Ingenuity Pathway Analysis  which eliminated all unmapped probe sets and which furthermore only allowed the probe set with the highest fold change for further analysis in the case that a single gene was represented by more than one probe set on the microarray.2 PCR Array System (PAHS-005E) containing 84 specific primer sets was used according to the manufacturer´s protocol . 1 μg total RNA was reverse-transcribed to cDNA with the RT2 First Strand Kit . The cDNA was combined with RT2 SYBR Green/ROX PCR master mix , and 10 µl of the mixture was added to each well of the PCR array (384-well format). The plates were run under conditions recommended by the supplier using the ABI-7900HT instrument .The Human Breast Cancer & Estrogen Receptor Signaling RTt method with an online analysis tool provided by the manufacturer of the PCR array  as described in detail elsewhere [−∆∆Ct method. Genes that achieved a p-value <0.05 were considered as statistically significant for the study.Data analysis was performed on four biological replicates per HBEC line using the ∆∆Clsewhere . Data weThe xCELLigence real-time cell analyzer (RTCA) system (Roche and ACEA Biosciences) is a label-free and non-invasive assay system based on a continuous measurement of electrical impedance to monitor biological processes of living cells in real time. The impedance is affected by the cells attached on the electrodes and gives information about their biological status, including cell number, viability, morphology and the degree of cell adhesion .Growth of the HBEC lines was continuously monitored for at least 24 h using 96-well plates that contain microelectrodes (E-plate 96) and the RTCA SP instrument . The measurement of the impedance background was performed with 100 µl of cell culture medium per well. After adding the cell suspension the final volume in a single well was 200 μL . Electric impedance was recorded every 30 min.Cell Index (CI), a relative and dimensionless value directly influenced by cell attachment, spreading and cell proliferation.The impedance is displayed as The neutral red cytotoxicity assay was conducted as described in Buhrke et al. . Trypan The activity of caspase 3 and 7 was detected using the Caspase-Glo 3/7 Assay . The cells were seeded in white walled 96-well plates at a density of 15,000 cells per well and allowed to attach. Twenty-four hours after plating cells were treated in triplicate with 0.5 µM staurosporin and DMSO as solvent control (0.5 %). Caspase-3 and -7 activities were determined after 12 h after staurosporin treatment according to the manufacturer’s protocol. Caspase-Glo 3/7 reagent was added (25 µl/well), gently mixed, and incubated for 30 min. The assay is based on the cleavage of a proluminogenic substrate, containing a DEVD sequence, by the caspases 3 and 7 which generates a luminescent signal produced by luciferase. Luminescence, that is proportional to the amount of caspase activity present, was measured using an Infinite M200 PRO multimode reader .2 of M13SV1, R2-2, and R2-N1 cells, respectively, were seeded on a 1–2 mm solidified layer of Growth Factor Reduced Matrigel  according to the manufacturer’s protocol one modification. The original matrigel was diluted with PBS to a final concentration of 5 mg/ml. The cells were grown in MSU-1 medium (see above). The medium was replaced every 2–3 days.Approximately 2000 cells per cm® 488 F(ab’)2 Fragment of Goat Anti-Rabbit IgG (A-11070), Alexa Fluor® 546 F(ab’)2 Fragment of Goat Anti-Rabbit IgG (A-11070), Alexa Fluor® 488 F(ab’)2 fragment of goat anti-mouse IgG (A-11017) and Alexa Fluor® 546 F(ab’)2 fragment of goat anti-mouse IgG (A-11018).Light microscopic analysis of the HBEC cell lines was carried out using the Carl Zeiss inverse Axiovert 135 microscope . A confocal laser scanning microscope  was used to visualize the immunofluorescent stains of cells grown in 3D culture. All primary antibodies were purchased from Abcam : anti-CD24 (ab30350), anti-Cytokeratin 14 (ab51054), anti-Cytokeratin 18 (ab668), anti-MUC1 (ab28081), anti-Oct4 (ab19857), anti-active Caspase-3 (ab32042) and anti-Integrin alpha 6 (ab20142). All secondary antibodies were purchased from Life Technologies : Alexa Fluor® Orange Nucleic Acid Stain  for 30 min.The spheroids grown in 3D culture were fixed with 3.7 % paraformaldehyde  for 10 min at room temperature. After three washing steps (for 5 min each) with PBS containing 0.1 % Tween-20 (PBS-T), the spheroids were permeabilized by 0.2 % Triton X-100 for 10 min at room temperature. After another washing cycle, the spheroids were blocked by using 10 % bovine serum albumin for 15 min. Subsequently, the spheroids were incubated with the first antibody (dilution 1:100 in PBS) for 1 h at 37° C. Afterwards, the spheroids were washed again with PBS-T and then incubated with a secondary antibody (dilution 1:200 in PBS) for 20 min at 37° C. Unbound secondary antibody was removed by washing the cells three times for 5 min with PBS-T. To counterstain the nuclei and the cytoskeleton the spheroids were incubated with Alexa Fluor 488 Phalloidin  and SYTOXt-test was used, and p-values <0.05 were considered statistically significant. The neutral red cytotoxicity data as well as the caspase 3/7 activity results were evaluated by using Student’s t-test, and p < 0.05 was regarded as statistically significant.In the gene array experiment, statistically significant deregulated genes were identified by a one-way ANOVA test (p < 0.05). Evaluation of the gene array data with the IPA software yielded z-scores in addition to p-values with respect to the level of deregulation of molecular networks. The z-score indicates how many standard deviations the sample lies away from the mean. Networks with z-scores <−2 are regarded as significantly inactivated, and z-scores >2 indicate significant activation of the respective network. The statistical data analysis of the PCR array was performed by using the web portal software of SABiosciences. Student’s


Four novel human TPM1 gene RNA isoforms were discovered , which were not identified in adult and fetal human cardiac tissues. TPM1λ was the most frequent isoform expressed in the malignant breast cell lines, and it was absent in normal breast epithelial cell lines. By western blotting, we were unable to distinguish between TPM1γ, λ, and ν protein expression, which were the only TPM1 gene protein isoforms potentially expressed. Some malignant cell lines demonstrated increased or decreased expression of these isoforms relative to the normal breast cell lines. Stress fiber formation did not correlate with TPM1γ RNA expression but significantly and inversely correlated with TPM1δ and TPM1λ expression, respectively. The exact differences in expression of these novel isoforms and their functional properties in breast epithelial cells will require further study.Nine malignant breast epithelial cell lines and 3 normal breast cell lines were examined for stress fiber formation and expression of TPM1 isoform-specific RNAs and proteins. Stress fiber formation was strong (++++) in the normal cell lines and varied among the malignant cell lines (negative to +++). Although TPM1 Expre tissues  9]. TPM. TPMα–κ)c tissue . The forc tissue , 12. TPMmyopathy .β . TPM1 γ and δ proteins  are known to be incorporated into epithelial cell stress fibers. It has been suggested that their expression may be downmodulated in human breast cancers  (Sigma-Aldrich) for 30 mins and rinsed 3 times with standard salt. Cells were mounted in Mowiol (Sigma-Aldrich) with 2.5% G n-Propyl Gallate (Sigma-Aldrich). The specimens were sequentially activated with different wave lengths of light and images were collected with a Leica AF6000 Deconvolution Microscope. The images were then merged to make a composite picture.Cell cultures were fixed in 3% paraformaldehyde in PBS  for 15 minutes at room temperature. The fixed cells were rinsed twice with standard salt solution (0.1 M KCl), 0.001 M MgClμg of RNA in a total volume of 40 mL was used to synthesize cDNA with SuperScriptRII  and oligo-dT primers following the manufacturer's specifications. For each PCR 3 μL of cDNA was used and GAPDH house-keeping gene RNA and TPM1 RNA were amplified as previously described [Total cellular RNA and protein were prepared from the samples, as previously described . For RT-escribed . Amplifiescribed . Qualita E. coli, and the DNA was extracted using Qiagen mini-prep kit . The isolated DNA was sequenced . Each clone was sequenced twice in both directions.Amplified products were ligated and cloned into the TA cloning vector (Life Technologies) following our published protocol . PositivIn order to more accurately relatively quantify novel TPM1 isoforms found herein, we sequenced 14 colonies of cloned DNA amplified from each of the cell lines with the TPM1.Exon1a(+) and TPM1.Exon9b(−) primer pair. The relative frequency of each isoform in each sample was then calculated.6 cells of each cell line was extracted with 100 μL of cell extraction buffer (Life Technologies) containing 10 mM Tris, pH 7.4; 100 mM NaCl; 1 mM EDTA; 1 mM EGTA; 1 mM NaF; 20 mM Na4P2O7; 2 mM Na3VO4; 1% Triton X-100; 10% glycerol; 0.1% SDS; 0.5% deoxycholate. The cell extraction buffer was supplemented with 1 mM PMSF and protease inhibitor cocktail  following manufacturer specifications. The pellets were discarded and 10 μL of supernatant from each sample was used for subsequent western blot analyses following our published protocol [r) between TPM1γ, TPM1δ, or TPM1λ RNA and stress fiber formation were calculated and plotted using Microsoft Excel. The p values were calculated using the Pearson calculator.Protein from 10protocol . Primaryprotocol , and CH1λ–ξ) were discovered in the breast epithelial cell lines (ξ, had not been predicted. However, several other predicted isoforms were not found. λ and TPM1ν encode proteins with 287 amino acid residues, whereas TPM1μ and TPM1ξ encode proteins with 284 amino acid residues. Interestingly, both TPM1λ and TPM1ν transcripts do not have exon 9a but do have exon 9b. This eliminates a stop codon and encodes for an additional 16 amino acids that would not be present in any other TPM1 isoform other than TPM1θ (λ and TPM1ν are differentiated by the fact that the former has exon 2b and exon 6b, but the latter contains exon 2a and exon 6a (μ and TPM1ε contain exon 9a and exon 9b. The protein coding ends at exon 9a and the stop codon as well as 3′-UTR comes from exon 9b. The only differences between these two isoforms are in exon 6. TPM1μ contains exon 6a whereas TPM1ε has got exon 6b (α and TPM1κ RNA (Tables β RNA expression but not protein expression was detected (Figures γ and TPM1δ were the dominant RNA isoforms expressed, although no evidence for TPM1δ protein expression could be identified (Tables δ protein. The skeletal muscle 34 kD protein detected with TM311 is probably TPM3α. It is conceivable that TPM1γ and δ are running together in the 36 kD band. TPM1λ RNA was identified in most malignant breast cancer cell lines but not in the benign cell lines, while TPM1ξ RNA was identified only in the MCF 10A cell line (μ and TPM1ξ protein expression (λ and ν would only be detected by the TM311 antibody. However, because we do not know their exact size on SDS PAGE, we cannot speculate as to whether they were expressed or not. We would assume that they could be very close in size to TPM1γ. However, because TPM1λ has a few more amino acids than TPM1γ, it could be slightly higher on an SDS PAGE gel. As can be seen in Stress fibers containing both tropomyosin and actin filaments were identified in the cell lines to varying degrees ranging from negative to ++++ . Four nell lines  Table 3λ–ξ were an TPM1θ . TPM1λ a exon 6a . Both TP exon 6b . None ofpression . TPM1λ aγ, TPM1δ, or TPM1λ RNA expression among the normal and malignant breast cell lines. There was no significant correlation between TPM1γ RNA and stress fiber formation, while TPM1δ RNA expression and TPM1λ RNA expression demonstrated significant positive and inverse correlations, respectively, with stress fiber formation.The actin cytoskeleton in epithelial cells contains stress fibers comprised of actin microfilaments and various actin-binding proteins . They plγ and δ and TPM2β have been shown to be expressed in normal human breast epithelial cells and their protein products are incorporated into stress fibers [ε and TPM4τ have also been shown to be expressed [γ and δ and TPM2β isoforms has been observed in some but not all malignant human breast cell lines and primary breast carcinomas [β were found to be elevated in primary breast cancers that gave rise to lymph node metastases compared to those that did not [δ but not TPM1γ RNA had a positive correlation with stress fiber formation in the cell lines studied.The high molecular weight tropomyosin isoforms TPM1s fibers . The lowxpressed . Downregrcinomas , 28, 29. did not . The datλ and ν are expressed as proteins or not. We suspect that they could be running with TPM1γ on the gel. In fact, we were not able to detect TPM1δ, which others have shown to be expressed in human breast epithelial cells [γ on our gels. These uncertainties should be able to be resolved with further studies employing a 2D SDS PAGE approach or by developing antibodies against the unique amino acids of the carboxy terminus of TPM1λ and ν.Given the identification and/or prediction of an increasingly greater number of tropomyosin isoforms than known in the past, we have embarked on a systematic examination of various human cell types for novel isoform expression. Indeed, in the data presented herein, we have detected four TPM1 RNA isoforms not identified in human tissues before. With the reagents and one-dimensional SDS PAGE techniques utilized we are unable to say whether TPM1al cells . We alsoλ was upregulated in many of the malignant cell lines compared to the normal cell lines. This increased RNA expression had a strong, statistically significant, inverse correlation with stress fiber formation among the cell lines. Again, future studies are required to study these differences at the protein level and whether they can be detected in fresh normal and malignant breast epithelial cells. Structural studies examining the incorporation into stress fibers and cellular location of the novel TPM1 isoforms and functional studies regarding the impact of their over- and underexpression are also warranted.At the RNA level, there were varying degrees of expression of the novel TPM1 isoforms between the “normal” and malignant breast epithelial cell lines. Of particular interest was the fact that TPM1δ and TPM1λ RNA had significant positive and inverse correlations, respectively, with stress fiber formation among the cell lines. The data would indicate that tropomyosin control of breast epithelial growth may be more complex than previously thought.Four novel tropomyosin RNA isoforms were detected in human breast epithelial cell lines, with varying degrees of expression between benign and malignant cells. Their function remains to be determined. TPM1


Ras-driven sarcomas in mice.Rhabdomyosarcomas (RMS) are mesodermal malignancies with skeletal muscle differentiation. The most common oncogenic mutations in RMS are in the RAS pathway. This study sought to identify actionable gene targets in sarcomas by selective targeting of the molecular networks that support the growth of Mouse RMS tumors were induced by expression of oncogenic Kras(G12v) and disruption of CDKN2A (p16p19) using ex-vivo transduction and intramuscular injection of transduced mouse satellite cells. These sarcomas identified a cluster of genes upregulated in mouse sarcomas and human RMS compared to normal skeletal muscle. A customized shRNA proliferation screen was used to screen this gene cluster for actionable transcripts that reduced sarcoma cell proliferation. Target gene effects on sarcoma growth were evaluated in mouse and human RMS cell lines and xenografts.Kras; p16p19null sarcomas. ASNS silencing in mouse and human sarcoma cell lines reduced the percentage of S phase cells and impeded new polypeptide synthesis. These effects of ASNS silencing were reversed by exogenous supplementation with asparagine. Finally, genetic silencing of ASNS in mouse sarcoma cells combined with depletion of plasma asparagine inhibited tumor growth in vivo.Five immediately actionable proliferation-relevant gene targets were identified, and the anti-proliferative effects of 5 candidate chemicals, including asparaginase and amino sulfoximine , were validated in mouse and human RMS cell lines. Silencing of ASNS, an amidotransferase that converts aspartate into asparagine, produced the strongest inhibitory effect on the growth of mouse The generation of new protein mass by rapidly proliferating sarcoma cells requires adequate Asparagine availability. Asparagine reliance of sarcoma cells may represent an actionable, metabolic vulnerability with potential anti-RMS therapeutic value.