Developing chemotherapy resistant cell lines can help to identify markers of resistance. Instead of using a panel of highly heterogeneous cell lines, we assumed that truly robust and convergent pattern of resistance can be identified in multiple parallel engineered derivatives of only a few parental cell lines.Parallel cell populations were initiated for two breast cancer cell lines (MDA-MB-231 and MCF-7) and these were treated independently for 18 months with doxorubicin or paclitaxel. IC50 values against 4 chemotherapy agents were determined to measure cross-resistance. Chromosomal instability and karyotypic changes were determined by cytogenetics. TaqMan RT-PCR measurements were performed for resistance-candidate genes. Pgp activity was measured by FACS.All together 16 doxorubicin- and 13 paclitaxel-treated cell lines were developed showing 2–46 fold and 3–28 fold increase in resistance, respectively. The RT-PCR and FACS analyses confirmed changes in tubulin isofom composition, TOP2A and MVP expression and activity of transport pumps . Cytogenetics showed less chromosomes but more structural aberrations in the resistant cells.We surpassed previous studies by parallel developing a massive number of cell lines to investigate chemoresistance. While the heterogeneity caused evolution of multiple resistant clones with different resistance characteristics, the activation of only a few mechanisms were sufficient in one cell line to achieve resistance. DNA-bound anthracycline binds DNA topoisomerase II, inducing DNA cleavage in an ATP-dependent manner There are several alternative strategies to develop chemotherapy response predictors, none of them being ideal, each having its own advantages and disadvantages. Genome scale molecular profiling, such as microarray analysis of tumor biopsies has the potential of most faithfully characterizing the molecular changes associated with response to therapy in the primary tumors An apparent problem with this and earlier studies was the use of a panel of highly heterogeneous cell lines for the identification of the predictors. Developing resistant cell lines to a given agent and then comparing the resistant and the sensitive parent cell line may circumvent this problem. For example MET inhibitors were recently investigated by Qi et al In the present study we went one step further in the simulation of the evolution of chemotherapy resistance development in a cell culture by significantly increasing the number of parallel developed cell lines. We assumed that truly robust and relevant resistance mechanisms will emerge in multiple cell lines and a clinically relevant convergent pattern of resistance can be identified. Twenty-nine subpopulations of two breast cancer cell lines were separated and treated with increasing concentrations of doxorubicin and paclitaxel for 18 months. These cells were then investigated to explore whether known resistance mechanisms consistently emerge and therefore the same strategy could be used to identify novel mechanisms as well.3, 1% minimal essential vitamins, and 20 000 kIE/l trasylol in a humidified atmosphere containing 5% CO2 at 37°C.The human breast cancer cell lines MCF-7 and MDA-MB-231 were obtained from ATCC, they were cultured in Leibovitz L-15 medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen), 2.5 mg/l transferrin, 1.1 g/l NaHCOTen sub-populations of each cell line for each of the two drugs were separated. The new cell lines were generated parallel by treatment with gradually increasing concentration of doxorubicin  or paclitaxel . After each week, the confluence was assessed: for cells lines with confluence below 50%, the treatment was stopped, for confluence between 50–70%, the treatment was maintained and for those with a confluence over 70% a portion of the cells was frozen. After 3 weeks of growth at a specific dose of treatment, the concentration was increased. The increase was in 10 fold increments until we reached 0.1× the clinical dose, then 0.3× and 0.6× followed. Before drug concentration increase an MTT cell proliferation assay (Cell Proliferation Kit I (MTT) Roche, IN, USA) was performed to monitor sensitivity of the resistant cell lines. Additionally, vehicle-treated parental cell lines were kept in culture for the duration of the study and these were used as control cell lines.50 values were determined for cisplatin , doxorubicin, paclitaxel and 5-fluorouracil  using the MTT Cell Proliferation Kit  and GraphPad Prism software.Cross-resistance measurements were performed on the parental and the resistant cell lines. IChttp://www.appliedbiosystems.com, CA, USA). The genes were selected based on a literature search, to include genes correlated to doxorubicin and paclitaxel resistance, breast cancer hormone receptors and survival associated genes. The complete list of genes and TaqMan IDs are listed in Table S1. For data analysis the SDS 2.2 software was used. The extracted delta Ct values were normalized to the average of 3 different housepeeking genes . Spearman rank correlation was computed to compare the expression for the genes and the resistance against doxorubicin and paclitaxel. Step-up multiple testing correction was performed TaqMan real-time PCR was used to measure the expression of selected genes using an Applied Biosystems Micro Fluidic Card System in 31 samples . The measurements were performed using an ABI PRISM® 7900HT Sequence Detection System as described in the products User Guide  were added to the cells and incubated for 0.5 h, then rhodamine 123 (10 µM) was added to the cells and after incubation the cells were collected and washed with fresh medium and resuspended in normal growth medium without doxorubicin/paclitaxel or rhodamine 123. Then the cells were centrifuged and washed in ice-cold PBS. At last, the cells were resuspended in 1 ml PBS and analyzed by flow cytometry. Fluorescence was measured from 104 cells and cell-count was plotted against rhodamine intensity.The ability of the cells to carry out of Pgp-mediated efflux was assessed by using the Pgp substrate rhodamine 123. Cells were seeded in 6-well plates at a density of 5×10After the end of the development, treated and vehicle-treated cell lines were cultured for 3–5 days. When the monolayer cultures showed ∼80% confluence, they were incubated overnight with 0,02–0,06 µg/ml final concentration of Colcemid. After detaching the cells with 1× trypsin/EDTA, chromosomal preparations were made according to standard techniques that used 0,067 M KCl as hypotonic treatment and fixation in methanol/acetic acid. Chromosome analysis was performed on metaphase cells G-banded with trypsin and Wright Giemsa stain. Ten metaphases were evaluated for each sample with an Axioskop 2 Mot Plus microscope  and Cytovision 3.6 or Mac Ktype 5.6 computer analysis system for karyotyping . Karyotypes were described according to the International System for Human Cytogenetic Nomenclature (ISCN 2009).pi is the frequency of a given number of a chromosome (i) in the cell lines. Thus, H was used to estimate the degree of numerical chromosomal heterogeneity within each of the developed resistant cell lines. Cell lines having an index at least 2.1 were designated as CIN positive (CIN+) tumors and remaining cell lines were designated as CIN negative (CIN−) tumors.Chromosomal instability (CIN) was measured by counting the average number of variations of chromosomes 3, 17 and 21 in ten different metaphases. Then, the Shannon Diversity Index (H) was computed using the formula At the end we obtained 29 resistant cell lines as a result of 18 months of treatment. There are 10 doxorubicin and 4 paclitaxel resistant MCF-7 cell lines, 6 doxorubicin and 9 paclitaxel resistant MDA-MB-231 cell lines. The relative resistance values compared to the original cell lines show up to 46 (average: 18.5) fold resistance against doxoxubicin and up to 28 (average: 15.4) fold resistance against paclitaxel. No cell line exhibited less than two-fold increase in the IC50 values. We determined the level of cross-resistance by determining the IC50 values against four widely used agents in the treatment of breast cancer . Although some of the cell lines exhibited significantly increased resistance against other agents as well, no significant correlation between the relative resistance levels was observed. The smallest relative resistance developed against cisplatin. Four cell lines (three of them treated with paclitaxel and one treated with doxorubicin) developed dramatic resistance against 5-fluorouracil. Gene expression of a set of selected genes was measured by TaqMan real-time PCR. The gene expression levels were correlated to the IC50 values using Spearman's rank correlation test. In the doxorubicin resistant cell lines the expression of TOP2A (p = 0.003) and two tubulin isoforms  was correlated to the level of resistance significantly. In the paclitaxel resistant cell lines, the expression of MVP (p = 0.009), of four tubulin isoforms  and of MAP4 (p = 0.001) correlated to resistance. ABCB1 reached a p value of 0.03 and 0.07 in the doxorubicin- and pacalitaxel-treated cell lines, respectively but these were not significant after multiple testing corrections. Hierarchical cluster visualizing the results is presented on The ability to export drugs via Pgp-activity was assessed by FACS using the Pgp substrate rhodamine 123. The originally paclitaxel-resistant MCF-7-R20 cell lines showed the highest cross-resistance against doxorubicin. The paclitaxel-resistant MDA-MB-231-R19 showed the highest resistance against paclitaxel and also showed rhodamine efflux. Two paclitaxel-resistant cell lines (MCF-6-R5 and MCF-7-R7) showed also increased resistance against doxorubicin and cisplatin. However, a general correlation between rhodamine efflux and multidrug resistance for other agents and other cell lines could not be observed. The results of the FACS analysis for selected sets of cell lines are shown in Cytogenetics was performed on all cell lines derived from the MDA-MB-231 cell line. The comparison of vehicle-treated and parental cell lines did not show new structural abnormalities, but the number of a few chromosomes  changed in the vehicle treated cells compared to the parental cells. Meanwhile, there was a high number of genetic changes in the generated cell lines. The new cell lines had 60–110 chromosomes, chromosomes 1, 17 and 21 had the most copies. Based on G-band staining, the highest variability among the chromosomes were observed on chromosomes 3, 7, 17, 20 and 21. Most stable chromosomes were X, 10, 13 and 16. The most common gains were on chromosomes 15, 18 and 21. The most common deletions were 9p21 and 18q21. Two of the cell lines (MDA-MB-231-R5 and MDA-MB-231-R11) differed from the other cell lines by having a nearly tetraploid modal chromosome number. The chromosomal changes and a representative karyotypes are depicted in Statistical measures of diversity typically integrate both number and abundance of chromosomes. The Shannon diversity index is preferable to other diversity measures like the Simpson's index as it is not dominated by the most frequent chromosome Starting from two breast cancer cell lines, one estrogen receptor positive and one estrogen receptor negative, using increasing concentrations of paclitaxel and doxorubicin we parallel developed 29 cell lines to establish a model similar to the evolution of the acquired drug resistance. We established a significantly higher number of resistant cell lines compared to previous studies In our study we focused on two questions. First, whether the multiple cell line-panel can re-identify previously clinically validated predictors of chemotherapy resistance. Our results suggest an affirmative answer and therefore validate the multiple cell line panel as a promising tool for the identification of clinically relevant biomarkers.Second, we were interested to see how often we see multiple drug resistance emerging when selected only with a single agent. Of the 29 resistant cell lines, 25 displayed at least two-fold increased resistance against another agent, 12 of the cell lines developed over two-fold resistance against at least three agents simultaneously, and two cell lines developed at least two-fold resistance against all four agents. Therefore, while most cell lines developed resistance to at least one more drug in addition to the agent used for selection pressure, the emergence of a truly multidrug resistance phenotype seems to be a relatively rare event. This is in line with the clinical observations that tumors that developed resistance to first line therapy are still often sensitive to second or third line agents In previous studies It is also notable that two genes selected for their previously described association with taxane resistance (TUBB2C ad TUBB3) were associated with doxorubicin resistance in our cell line panel, but with the opposite trend, and MVP, selected for its reported association with doxorubicin resistance, showed a significant correlation with taxane resistance.Our study shows that the heterogeneity results in the evolution of multiple drug resistant tumor cell populations with different geno- and phenotype. The evolution of multiple resistant cell populations may require developing new treatment strategies to prevent or overcome resistance. Multiple samples from the tumor may give better insight into the clonal composition of the tumor. However it is impractical and not cost-efficient in clinical practice to perform multiple predictive tests from the same tumor, but examination of the extent of heterogeneity from a single sample may help to guide treatment decisions Finally, we must note a limitation of our cell line models, i.e. they do not represent the tumor completely as they do not take into consideration the interactions between the tumor epithelial cells and the tumor microenvironment. Meanwhile, the tumor microenvironment has been shown to be an important player in response/resistance to chemotherapy In summary, we aimed to model in vitro the emergence of drug resistance in cancer and were able to distinguish several previously clinically validated predictors. In contrast to our expectations chemoresistance evolution can emerge by only a limited number of independent mechanisms.Table S1(DOCX)Click here for additional data file.


Ferula species are reputed in folk medicine for the treatment of a variety of disorders. There have been sporadic reports on the chemopreventive and chemosensitizing activities of some terpenoid coumarin derivatives from the genus Ferula. The present study investigated the cytotoxic activity of 11 phytochemicals  from Ferula species together with a newly synthesized prenylated derivative of curcumin (gercumin II).Cytotoxic activity of phytochemicals was evaluated against ovarian carcinoma (CH1), lung cancer (A549) and melanoma (SK-MEL-28) cell lines using MTT assay.50 values in the micromolar range. The highest activity against CH1 and A549 lines was from conferone while stylosin and tschimgine were the most potent compounds against SK-MEL-28 line. In conclusion, the findings of the present investigation did not support a potent cytotoxic activity of the tested phytochemicals against CH1, A549 and SK-MEL-28 cell lines. With respect to previous reports, the beneficial impact of these phytochemicals in cancer therapy may be more attributable to their chemopreventive or chemosensitizing activity rather than direct cytotoxic effects.Overall, moderate cytotoxic activity was observed from the tested compounds with IC During recent decades, there has been an increasing demand for finding newer and safer chemotherapeutic agents. Natural products, in particular plants, have long been used for their medicinal properties in several traditional systems of medicine. Over 60% of current cytotoxic agents have been derived from natural sources including plants, marine organisms and microorganisms, either directly or by chemical synthesis based on natural lead compounds ,2. ThereFerula (Apiaceae) comprises about 170 species, of which 30 have been included in Iranian flora and some are endemic. Plants belonging to this genus are distributed throughout central Asia, Mediterranean region and Northern Africa, and are well reputed in traditional medicine for the treatment of a variety of disorders , and those giving similar spots were combined, solvent evaporated and crystallized. Gercumin II was obtained as yellow crystals . The chemical structure of gercumin II was confirmed by 2 culture flasks (Iwaki/Asahi Technoglass) as adherent monolayer cultures in Minimal Essential Medium (MEM) supplemented with 10% heat-inactivated fetal bovine serum, 1 mM sodium pyruvate, and 2 mM L-glutamine  without antibiotics. Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2 and 95% air.CH1  cells were donated by Lloyd R. Kelland . A549  and SK-MEL-28  cells were kindly provided by Brigitte Marian  and Christoph Hoeller . Cells were grown in 75 cm3 (CH1), 4.0 × 103 (A549), and 4.0 × 103 (SK-MEL-28), to ensure exponential growth of untreated cells. Cells were allowed to settle and resume exponential growth in drug-free complete culture medium for 24 h, followed by addition of dilutions of the test compounds in 100 μL/well of the same medium. Test compounds were used from fresh stock solutions in dimethylsulfoxide (DMSO) and diluted in media or buffer as appropriate to a maximum of 0.3% DMSO. After continuous exposure for 96 h, the medium was replaced by 100 μL/well RPMI 1640 medium  plus 20 μL/well solution of MTT in phosphate-buffered saline (5 mg/mL) . After incubation for 4 h, medium/MTT mixtures were removed, and the formazan product formed by viable cells was dissolved in DMSO (150 μL/well). Optical densities were measured at 550 nm with a microplate reader (Tecan Spectra Classic), using a reference wavelength of 690 nm to correct for non-specific absorption. The quantity of viable cells was expressed as percentage of untreated controls, and 50% inhibitory concentrations (IC50) were calculated from concentration-effect curves by interpolation. Evaluation was based on means from three independent experiments, each comprising six replicates per concentration level.Cytotoxic activity in the cell lines mentioned above was determined by the colorimetric MTT assay -2,5-diphenyl-2H-tetrazolium bromide, purchased from Fluka). Cells were harvested from culture flasks by trypsinization and seeded in 100 μL aliquots in MEM supplemented with 10% heat-inactivated fetal bovine serum, 1 mM sodium pyruvate, 2 mML-glutamine, and 1% non-essential amino acids (100x) into 96-well microculture plates (Iwaki/Asahi Technoglass). Cell densities per well were adjusted to 1.5 × 1050 values mostly in the micromolar range. IC50 values are listed in Table Cytotoxic activity of the phytochemicals was studied by means of a colorimetric microculture assay (MTT assay) in three human cancer cell lines representing different tumor entities: ovarian carcinoma CH1), lung cancer (A549) and melanoma SK-MEL-28) yielding IC, lung ca8 yieldin50 values for all evaluated compounds were markedly higher than that of the reference standard, cisplatin. In addition, no obvious structure-activity relationship was found for the tested compounds. Overall, conferone exhibited the highest activity among the tested compounds against A549 and CH1 cell lines. In previous investigations, Barthomeuf and colleagues demonstrated that conferone enhances the cytotoxic activity of vinblastine in MDCK-MDR1 cells. This activity was shown to be due to the potent inhibition of P-glycoprotein by this phytochemical and consequent reversal of multidrug resistance [in vitro and in vivo chemopreventive as well as in vitro anti-tumor properties [et al. did not found any significant activity of isolated sesquiterpene coumarins from F. gummosa against M14, MCF-7, T98G, A549, Saos-2, FRO, and U937 cell lines. The only exception was the cytotoxic effect of feselol against the U937 cell line [To our knowledge, data regarding cytotoxic activity of the compounds tested herein have been very few and for some of them lacking. All tested compounds displayed cytotoxic activity against the tumor cell lines. However, the obtained ICsistance . The samsistance . Howeversistance ,18. The sistance ,20. As foperties -23. The operties . Moreoveoperties . Neverthoperties . Galbanioperties . Finallyoperties . In accoell line . Anotherell line -34. It aell line . GercumiFerula extracts as these extracts are known to possess antioxidant properties which is mainly due to their phenolic constituents [In summary, the findings of the present investigation did not support a potent cytotoxic activity of the tested phytochemicals against CH1, A549 and SK-MEL-28 cell lines. Lack of remarkable anti-tumor activity of the tested compounds in the present study, which is in contrast with some previous findings, might be attributed to the possible differential effects of these phytochemicals against different cell lines. Besides, since antioxidants have a dual role in fighting cancer , it rematituents . FinallyNMR spectral data of tested compounds are available as supporting information.The authors have no conflict of interests to declare.AS and SMV conceived the study and designed the experiments. MI and SMV performed the experimental work including phytochemical and cell culture investigations, respectively. AS and SMV were involved in data interpretation and drafting the manuscript. All authors read and approved the final manuscript.1H-NMR spectrum of umbelliprenin.Click here for file1H-NMR spectrum of 7-isopentenyloxycoumarin.Click here for file1H-NMR spectrum of herniarin.Click here for file1H-NMR spectrum of stylosin. Part A.Click here for file1H-NMR spectrum of stylosin. Part B.Click here for file1H-NMR spectrum of stylosin. Part C.Click here for file1H-NMR spectrum of tschimgine. Part A.Click here for file1H-NMR spectrum of tschimgine. Part B.Click here for file1H-NMR spectrum of galbanic acid.Click here for file1H-NMR spectrum of farnesiferol A.Click here for file1H-NMR spectrum of diversin.Click here for file1H-NMR spectrum of conferone.Click here for fileH-NMR spectrum of acantrifoside E. Part A.Click here for file1H-NMR spectrum of acantrifoside E. Part B.Click here for file1H-NMR spectrum of acantrifoside E. Part C.Click here for file1H-NMR spectrum of mogoltadone.Click here for file1H-NMR spectrum of gercumin II.Click here for file


Activating mutations of FMS-like tyrosine kinase-3 (FLT3) are found in approximately 30% of patients with acute myeloid leukemia (AML). FLT3 is therefore an attractive drug target. However, the molecular mechanisms by which FLT3 mutations lead to cell transformation in AML remain unclear. To develop a better understanding of FLT3 signaling as well as its downstream effectors, we performed detailed phosphoproteomic analysis of FLT3 signaling in human leukemia cells. We identified over 1000 tyrosine phosphorylation sites from about 750 proteins in both AML (wild type and mutant FLT3) and B cell acute lymphoblastic leukemia  cell lines. Furthermore, using stable isotope labeling by amino acids in cell culture (SILAC), we were able to quantified over 400 phosphorylation sites  that were responsive to FLT3 inhibition in FLT3 driven human leukemia cell lines. We also extended this phosphoproteomic analysis on bone marrow from primary AML patient samples, and identify over 200 tyrosine and 800 serine/threonine phosphorylation sites in vivo. This study showed that oncogenic FLT3 regulates proteins involving diverse cellular processes and affects multiple signaling pathways in human leukemia that we previously appreciated, such as Fc epsilon RI-mediated signaling, BCR, and CD40 signaling pathways. It provides a valuable resource for investigation of oncogenic FLT3 signaling in human leukemia. FMS-like tyrosine kinase-3 (FLT3) is a class III receptor tyrosine kinase (RTK) that also includes C-KIT, C-FMS, and platelet-derived growth factor receptor (PDGFR). Type III RTKs share a common structure consisting of 5 extracellular immunoglobulin-like domains, a single transmembrane domain, a cytoplasmic juxtamembrane region, and a cytoplasmic kinase domain interrupted by the kinase insert. FLT3 is primarily expressed in early myeloid and lymphoid progenitors and plays an important role in their proliferation and differentiation. In human acute leukemia, FLT3 is expressed on the surface of the leukemic cells in 70–90% acute myeloid leukemia (AML) patients and most B-acute lymphoblastic leukemia (B-ALL) MLL-rearranged acute lymphoblastic leukemias Overexpression and activating mutations of receptor tyrosine kinases (RTKs) are known to be involved in the pathogenesis of many types of cancer. Mutations in FLT3 are commonly detected in patients with AML. The most common of these mutations are internal tandem duplications (ITDs), which occur in 25–30% of these patients FLT3-ITDs activate many of the same signal transduction pathways as the native receptor, such as RAS-MAPK pathway, JAK-STAT pathway, PI3K, SRC, and phospholipase C-gamma To gain a global view of oncogenic FLT3 signaling in human leukemia cells, we applied PhosphoScan®, an immunoaffinity-MS profiling approach to leukemia cell lines containing activated FLT3 mutations (FLT3-ITD and amplification) www.phosphosite.org). Based on protein classification, cytoskeletal proteins, cell surface molecules, and adaptor and scaffold proteins are the most highly phosphorylated protein types , and analyzed by LC-MS/MS mass spectrometry (IAP-MS) in types . Moreovein types , consistin types . On the in types . Y630 isSince FLT3 is also involved in the pathogenesis of B cell acute lymphoblastic leukemia (B-ALL), we investigated FLT3 tyrosine signaling in three B-ALL cell lines, SEM, RS and REH. While SEM cells express high level of FLT3, RS and REH cells express relative low levels of wild type FLT3. In addition, SEM, but not RS and REH, is sensitive to FLT3 inhibitorTo find out a subset of phosphorylated proteins and their respective sites that are regulated by FLT3, we employed stable isotope labeling with amino acids in cell culture (SILAC) to differentially label proteins derived from FLT3 inhibited versus uninhibited leukemia cell lines. We used MLN518, a quinazoline-based inhibitor of the type III receptor tyrosine kinases FLT3, PDGFRA, CSF1R, and C-KITSome of the known FLT3 regulated phosphorylation proteins were significantly inhibited by MLN518, including FLT3 itself, and its downstream effectors, SHC, STAT5A, and ERK. In addition, we identified phosphorylation sites on adaptor proteins previous linked to wild type FLT3 signaling, such as GAB2, SHP-2, PLCG2, and SHIP1 In addition to FIT3-ITD AML cell lines, we identified a total of 160 MLN518 sensitive sites from 120 proteins in SEM cells, significantly extended our knowledge of FLT3 signaling in ALL. Adaptor/scaffold, cytoskeletal proteins, cell surface molecules, and adhesion/extracellular matrix proteins make up the majority of protein types . SimilarSer424 was further confirmed by Western blot with phospho-specific antibody . ERF, an ets domain transcriptional repressor, serves as a link between RAS/MAPK pathway and cell cycle progression Thr526 by ERK renders it unable to repress c-myc and cdc2 expression Because FLT3 receptor signaling engages the MAPK pathway and also regulated the cell cycle antibody , locatinTo understand the role of FLT3 activation in primary patients, we profiled bone marrow cells from AML samples with known FLT3 mutational status. We identified 209 tyrosine phosphorylation sites from over 200 proteins in 6 AML patients, three of which contains FLT3-ITD mutation  and S2. Ser424, NFkB-p105Ser907, AMLSer249,276,Thr273, and ERFThr526. Overall, we identified 70 drug sensitive serine/threonine phosphorylated proteins  in 3 FLT3-ITD patients  cells, as well as Y266 and 476 of GAB2 in Molm 14 cells. Our data also showed that phosphorylation of different sites within the same protein can also be differentially regulated. For example, Y383 and Y598, but not Y208 on GIT1 (G protein-coupled receptor kinase interactor 1) are responsive to MLN518 In MV. A similar observation was made for tyrosine sites on ENO1 and HCK. This highlights the importance of measuring the degree of site-specific phosphorylation rather than phosphorylation the protein as a whole in order to obtain an accurate picture of a protein's function.In SEM cells, we observed multiple FLT3 regulated sites on key elements of B-cell receptor signaling pathways such as BCAP, CD19, LAB, DOK1, GAB1, and GAB2. In addition, we confirmed phosphorylation of BCAP is regulated by FLT3. BCAP binds PI3K and plays an essential role in B cell development In this study, we identified five FLT3 tyrosine phosphorylation sites in primary leukemia cell lines, adding new information on how phosphorylation may affect the activity of wild-type FLT3 and FLT3-ITD. FLT3 has been implicated in the pathogenesis of both AML and B-ALL. We observed that in both AML and B-ALL cell lines, FLT3 activates many of the similar signaling pathways, such as JAK-STAT pathway, RAS-MAPK pathway, and phospholipase C-gamma  and 4. YWhen we compared phosphotyrosine profile between two FLT3-ITD cell lines  and Molm 14) and three FLT3-ITD AML patients, we noticed that 77% (130 out of 168) of phosphoproteins identified in AML patients were also present in these two cell lines, suggesting that FLT3-ITD cell lines are good models to understand FLT3-ITD signaling for primary patients . On the Several small molecule FLT3 inhibitors are in phase I/II clinical trials. However, the fundamental relationship between FLT3 inhibition, its consequences on downstream signaling, and the onset of cytotoxicity remains poorly understood ueous One solution cell proliferation assay (Promega). Collection and use of de novo AML patient bone marrow samples were approved by the human subjects Institutional Review Boards of Oregon Health & Science University and Portland VA Medical Center with written consent from patients. Patient information was not revealed in this study, and the data were analyzed anonymously.8 human AML cell lines , Molm 14, Marimo, Me-F2, KY821, OCI/AML3, Nomo-1, and ML-1) and 3 human B-ALL cell lines , and REH) were included in mass spectrometry analysis. Both MV and Molm 14 contain FLT3-ITD mutation. SEM contains amplified copy number of FLT3. Detailed history about these cell lines were provided in 8 cells (or 1×108 bone marrow cells) were lysed in urea buffer, trypsin digested lysate were purified by Sep-pak C18 column. Then, lyophilized peptides were redissolved and immunoaffinity purified with pY-100 antibody, which interacts with a broad range of tyrosine-phosphorylated peptides. pTyr-containing peptides were concentrated on reverse-phase micro tips. Supernatants after pY-100 immunoprecipitation were further immunoaffinity purified with PXSP and PXTP (Phospho-MAPK/CDK substrate) motif antibodies, which detect phospho-serine in a PXS*P or phospho-threonine in a PXT*P motif. Tandem mass spectra (ms/ms) were collected in a data-dependent fashion with an LTQ ion trap mass spectrometer or and LTQ-Orbitrap mass spectrometer (ThermoFinnigan) in which the top 10 ions were selected for ms/ms. Sequest (Thermo Fisher Scientific) searches were done against the NCBI human database released on July 02, 2009, , allowing for phosphorylation (STY+80) and oxidized methionine (M+16) as differential modifications using a precursor tolerance of 1.2 Da for LTQ data or 50 ppm LTQ Orbitrap data. LTQ data was searched fully tryptic, while Orbitrap data was searched with partial tryptic specificity. The PeptideProphet probability threshold was chosen to give a false positive rate of 1% for the peptide identifications . Briefly, about 2×10ications  [49]. Ph8 cells from either heavy or light cultures were combined and carried through the phosphopeptide immunoprecipitation protocol. Methods for LTQ-FT MS, Sequest searches and Vista  were described previously MV, Molm 14, SEM, and K562 cells were grown in RPMI-1640 (lacking arginine and lysine) supplemented with 10% dialyzed fetal bovine serum, and L-lysine/HCl and L-arginine/HCl  for light cultures or L-arginine/HCl  and L-lysine:2 HCl   for heavy cultures, as previously described We used the following statistical and computational tools from Gene Set Enrichment Analysis  for Molecular Signature Database analysis; BIOBASE Knowledge Library for Gene Ontology analysis. STRING 8.2 for potential protein protein interaction analysis Cells were lysed in 1× cell lysis buffer  supplemented with Protease Arrest™ (G Biosciences) and separated by electrophoresis. All antibodies and reagents for immunoblotting were from Cell Signaling Technology, Inc.Figure S1FLT3-ITD cell lines are sensitive to its inhibitor. (A) Sensitivity of AML and B-ALL cell lines to FLT3 inhibitor (MLN518). % control means percent of viable cells after drug treatment as compared to untreated control. (B) Phosphorylation and expression of CSF1R, C-KIT, and PDGFRA in leukemia cell lines. GDM-1, M-07e, and H1703 cell lines are positive controls for the expression of CSF1R, C-KIT, and PDGFRA, respectively. (C) Phosphorylation of p70S6 kinase is inhibited by FLT3 inhibitor.(TIF)Click here for additional data file.Figure S2Western blot analysis confirms MLN518 responsive pTyr-sites in AML and B-ALL cell lines by Sorafenib treatment. (A), (B), and (C) Immunoblotting of MV, Molm 14, and SEM cells treated with 100 nM FLT3 inhibitor (sorafenib) for 2 hours with different phospho-specific antibodies. K562 cells were included as controls.(TIF)Click here for additional data file.Figure S3Venn diagram showed the overlap of tyrosine phosphorylated proteins identified between two FLT3-ITD AML cell lines and three FLT3-ITD primary AML patients.(TIF)Click here for additional data file.Table S1Phosphopeptides identified by LC-MS/MS in AML and B-ALL cell lines, as well as primary AML patients. P1–P3 are FLT3 wild type AML patients, P4–P6 are FLT3-ITD AML patients. ‘Y*’, ‘S*’/, and ‘T*’ in peptide indicates phosphorylated tyrosine, serine, or threonine residue, respectively. Spectra links were provided, as well as other important mass spectrometry parameters . False discovery rate (fdr) for each dataset was also listed.(XLS)Click here for additional data file.Table S2Non-redundant phosphopeptides identified by LC-MS/MS in this study presented in a format that is based on individual cell line or AML patient. ‘y’, ‘s’, and ‘t’ in peptide indicates phosphorylated tyrosine, serine, or threonine residue, respectively. Data from duplicate experiments were combined. Tyrosine kinases profile in AML cell lines and patients were summarized as well. ‘§’ indicates known phosphorylation site.(XLS)Click here for additional data file.Table S3Phosphotyrosine SILAC data of MV, Molm 14, SEM, and K562 cell lines, and phosphoserine (PXSP) and phosphothreonine (PXTP) SILAC data of MV, SEM, and K562 cell lines, treated with FLT3 inhibitor (MLN518). ‘Y*’ ‘s’, and ‘t’ in peptide indicates phosphorylated tyrosine, serine, or threonine residue, respectively. ‘§’ indicates known phosphorylation. Results from duplicate run were combined.(XLS)Click here for additional data file.Table S4List of proteins and their protein types identified in SILAC study of FLT3-ITD cell lines (> = 2 fold) that were also present in three FIT3-ITD primary patients. Both tyrosine phosphorylated and serine/threonine phosphorylated proteins were listed.(XLS)Click here for additional data file.


Chromogranin A is a neuroendocrine secretory product and its loss is a feature of malignant NEN de-differentiation. We hypothesized that chromogranin A fragments were differentially expressed during NEN metastasis and played a role in the regulation of NEN proliferation.Chromogranin A mRNA (PCR) and protein (ELISA/western blot) were studied in 10 normal human mucosa, 5 enterochromaffin cell preparations, 26 small intestinal NEN primaries and 9 liver metastases. Cell viability (WST-1 assay), proliferation (bromodeoxyuridine ELISA) and expression of AKT/AKT-P (CASE ELISA/western blot) in response to chromogranin A silencing, inhibition of prohormone convertase and mTOR inhibition (RAD001/AKT antisense) as well as different chromogranin A fragments were examined in 4 SI-NEN cell lines.p<0.0001) in small intestinal NENs compared to normal enterochromaffin cells. Western blot identified chromogranin A-associated processing bands including vasostatin in small intestinal NENs as well as up-regulated expression of prohormone convertase in metastases. Proliferation in small intestinal NEN cell lines was decreased by silencing chromogranin A as well as by inhibition of prohormone convertase (p<0.05). This inhibition also decreased secretion of chromogranin A (p<0.05) and 5-HT (p<0.05) as well as expression of vasostatin. Metastatic small intestinal NEN cell lines were stimulated  and AKT phosphorylated (Ser473: p<0.05) by vasostatin I, which was completely reversed by RAD001 (p<0.01) and AKT antisense (p<0.05) while chromostatin inhibited proliferation .Chromogranin A mRNA and protein levels were increased (37-340 fold, Chromogranin A was differentially regulated in primary and metastatic small intestinal NENs and cell lines. Chromogranin A fragments regulated metastatic small intestinal NEN proliferation via the AKT pathway indicating that CgA plays a far more complex role in the biology of these tumors than previously considered. Chromogranin A (CgA), a heat stable, hydrophilic acidic protein of ~460 amino acids, is a member of the granin family of secretory proteins that are ubiquitous to the nervous, endocrine and immune system ,2. It foWestern blot identifies a range of CgA fragment sizes from ~9-85 kDa depending on processing, neuroendocrine cell type and antibody used , but theAlthough CgA is a well-characterized product of NENs, very few studies have investigated the role of either CgA or its cleavage fragments as regulators of small intestinal NEN (SI-NEN) proliferation, the commonest tumor in this class ,18. Givein vitro. We also examined the effect of CgA silencing and pharmacologic inhibition of prohormone convertase on tumor cell proliferation, secretion and post-transcriptional changes in CgA fragment expression. The results identified a role for CgA peptides in the regulation of NEN proliferation at the level of AKT signaling. Targeting AKT or prohormone convertases specifically decreased proliferation, especially in metastases.We specifically focused on SI-NENs since these are common and there are a number of well-characterized cell lines available -24. Initn=26), now classified as G1 NETs  as well as compared to EC cell preparations (p<0.01) (We next examined CgA protein expression in these samples using ELISA 2C. The (p<0.01) 2C. Protdata not shown) were highly expressed in metastases. Other processing fragments as well as fragments that included pancreastatin were also identified in neoplasia; levels were increased in comparison to normal EC cells . CgA was elevated, particularly in P-STS, compared to H-STS (p<0.001) and L-STS cells (p<0.01); CgA, likewise, was higher in KRJ-1 compared to H-STS cells  was also elevated compared to normal EC cells (3-6000 fold), while the two metastatic cell lines (L-STS and H-STS) expressed lower levels of CgA mRNA in comparison to normal EC cells (0.08-0.3 fold). Both primary cell lines expressed higher levels of CgA mRNA than the metastatic cell lines  and were particularly higher in the primary (P-STS) than in its metastasis  (CgA protein levels (measured by ELISA) also differed in the SI-NEN cell lines  2D. The  p<0.05) 2B. The four cell lines all expressed detectable parent CgA bands but expression appeared higher in primary cell lines 2F. SincPCSK1, PC1-3, respectively) was investigated in normal human mucosa, EC cell preparations, localized SI-NENs, primaries with metastasis and liver metastases. Levels were over-expressed in SI-NENs (p<0.001) and were increased in metastases compared to localized tumors (p<0.05) suggesting this enzyme may be a relevant regulator of CgA processing in SI-NEN metastases.Given the evidence for different CgA peptide fragments, mRNA and protein expression of the CgA processing enzyme prohormone convertase 1  phase ,21,24. Adata not shown), which resulted in a significant decrease in proliferation  and 5-HT . These physiological alterations, a decrease in CgA and 5-HT secretion, were associated with alterations in CgA processing following inhibition of prohormone convertase , while chromostatin had an anti-proliferative effect on the primary cell line P-STS (p<0.02) in both metastatic cell lines (L-STS and H-STS) but had no effects on primary tumor cell lines (P-STS and KRJ-1)  (p<0.01). AKT antisense also was able to reverse vasostatin-mediated BrdU uptake (proliferation) (p=0.03)  . This efot: 25%) 5A, C aneration) 5E. In c(p=0.03) 5B as we(p=0.03) 5D in th(p=0.03) 5F. ThesChromogranin A is a pro-hormone that is differentially processed into peptides that regulate a range of biological functions including cell proliferation, angiogenesis and hormonal secretion . The curCgA mRNA and proteins/peptides were, in comparison to normal mucosa and EC cells, increased in SI-NENs, which were also characterized by the expression of CgA processing fragments consistent with N-terminal fragments. The mechanisms underlying the altered expressions may reflect either transcriptional or post-translational mechanisms. CgA transcription is well-known to be regulated via CRE sites in the promoter . We haveper se [While CgA is commonly considered a marker of tumor load and plasma levels are related to progression free survival in GEP-NENs ,39, thisper se ,41. In tper se , suggestKip1) [Kip1 [-4 to 10-7M [One of the CgA fragments that was differentially processed during SI-NEN metastasis was vasostatin I/II which is recognized to have vasoconstrictive effects on small and medium resistance vessels in cardiovascular system . AlthougKip1) . In the 1) [Kip1 . These v1) [Kip1 . These eto 10-7M . The twoto 10-7M , we postIn contrast to vasostatin, chromostatin inhibited proliferative activity in P-STS cells through inhibition of AKT phosphorylation, which is, to the best of our knowledge, the first identification that this CgA fragment has an anti-proliferative effect in NENs.PC1 and PC2 mRNA expression has been suggested to play distinct roles in the activation of brain pro-proteins, particularly CgA, while expression of this enzyme itself appears regulated at a CRE-level, at least in the pancreatic NEN cell line BON [PC1/2 mRNA was identifiable in normal EC cells, but was over-expressed in SI-NENs while PC1 itself was significantly elevated in metastases. Inhibiting this enzyme resulted in a decrease in H-STS proliferation as well as down-regulation of CgA and 5-HT secretion demonstrating the sensitivity of post-translational modifications within the neoplastic CgA system. An emerging area of interest is regulation of pro-hormone processing enzymes, either spatially or at the level of cellular expression, that may play an important role in cleavage and secretion of hormones . The claline BON . In the In conclusion, we identified that CgA was differentially regulated in primary and metastatic SI-NEN cell lines, which exhibited elevated  but different patterns of CgA fragments with different functions. Specifically, N-terminal fragments stimulated metastatic NEN cell line proliferation while middle fragments inhibit localized tumor cell proliferation via the AKT/mTOR pathway. The presence and over-expression of prohormone convertases 1-3 in neuroendocrine neoplasms suggested that CgA was altered at a post-translational stage via this enzymes. It therefore seems likely that the CgA fragment pattern may be of value in evaluating the biologic behavior of NENs and the region-specific antibodies may be of potential use when seeking to identify individual malignant tumor phenotypes.


Hantavirus. Furthermore, reservoir host species are increasingly recognized besides representatives of the order Rodentia, now including members of the mammalian orders Soricomorpha/Eulipotyphla and Chiroptera. Despite the great interest created by emerging zoonotic viruses, there is still a gross lack of in vitro models, which reflect the exclusive host adaptation of most zoonotic viruses. The usually narrow host range and genetic diversity of hantaviruses make them an exciting candidate for studying virus-host interactions on a cellular level. To do so, well-characterized reservoir cell lines covering a wide range of bat, insectivore and rodent species are essential. Most currently available cell culture models display a heterologous virus-host relationship and are therefore only of limited value. Here, we review the recently established approaches to generate reservoir-derived cell culture models for the in vitro study of virus-host interactions. These successfully used model systems almost exclusively originate from bats and bat-borne viruses other than hantaviruses. Therefore we propose a parallel approach for research on rodent- and insectivore-borne hantaviruses, taking the generation of novel rodent and insectivore cell lines from wildlife species into account. These cell lines would be also valuable for studies on further rodent-borne viruses, such as orthopox- and arenaviruses.Due to novel, improved and high-throughput detection methods, there is a plethora of newly identified viruses within the genus  Emerging zoonotic viruses have received tremendous interest within recent years and are perceived as a major health risk for humans ,2. AmongHantavirus within the family Bunyaviridae [Bunyaviridae, hantaviruses are harbored by small mammals, mainly rodents (order Rodentia). In general, each hantavirus species is thought to be carried and transmitted to humans by a single reservoir species. Thus, the prototype hantavirus species, the Hantaan virus, was exclusively detected in the striped field mouse Apodemus agrarius in Asia [Myodes (Clethrionomys) glareolus [Apodemus species [Microtus arvalis representing the reservoir host, but was also molecularly detected in other vole species [Hantaviruses are enveloped viruses with a segmented RNA genome of negative polarity. Taxonomically, these viruses belong to the genus aviridae . In cont in Asia . Similarlareolus ,21. For  species . In addi species ,24,25. T species .Recently, a large number of shrews and moles (order Soricomorpha/Eulipotyphla) and bats (order Chiroptera) have been identified as reservoirs of additional hantaviruses ,30,31,32In contrast, in the reservoir host a hantavirus infection is usually believed to be persistent and not associated with (at least obvious) disease. However, field studies in bank voles indicated that hantavirus infection has a negative impact on over-winter survival, and histopathologic lesions in several organs have been observed in hantavirus-infected white-footed mice and in deer mice ,49,50. TCorona- or Paramyxoviridae. These research questions necessitate, however, novel tools and methods that allow comparative infection studies and immunological evaluation of virus-host interactions among a broad species context, not only including wildlife, but also livestock and companion animal species.With the discovery of hantaviruses in a broad spectrum of reservoirs and the identification of unexpected spillover and putative host-switch events, reservoir-host centered infection models become of immediate interest ,51. In ain vitro model systems do not reflect characteristics of reservoir specific virus-host interactions [in vivo data on the immunology of hantavirus infection in their natural reservoir stem from animal experiments on Norway rats, deer mice, bank voles and cotton rats . Howevein vitro models such as bat cell lines. The generation of these cell lines across a broad range of species has already provided important insights into virus-host interaction on a cellular level and on innate immune functions, e.g., interferon response [Among all mammalian reservoir hosts, bats are most likely the order of mammals that has received the most attention from the virological research community in recent years ,55,56. Aresponse ,61,62,63in vitro models to study hantaviruses.The many similarities between bat-, insectivore- and rodent-borne viruses could stimulate a synergistic approach for reservoir host-derived Cell culture is the mainstay of classical virology and even in times of highly sensitive and high-throughput detection methods, isolation of a virus in cell culture enables its thorough phenotypical characterization. While in the early era of virology, isolation by cell culture was routinely performed; it has become something of an outdated method for many years. Cell lines such as Vero cells, derived from the kidney of an African green monkey, are widely used for virus isolation and especially the subclone Vero E6 provides an excellent environment for RNA viruses to replicate due to an impaired IFN response ,65.Pteropus alecto), the species which naturally harbors this virus [et al. showed isolation and replication of a bat herpesvirus derived from the bat Miniopterus schreibersii in primary bat cells after unsuccessful isolation attempts in 14 other mammalian cell lines [Vero E6 cells have enabled the isolation of a variety of hantaviruses ,68,69,70is virus . In thisis virus . In addill lines .i.e., members of the family Coronaviridae), the number of hantavirus isolates of reservoir or human origin is still rather small [Although hantaviruses are not as hard to isolate as other zoonotic viruses  for multiple passages, suggesting an origin of the virus in bats [On the other hand, reservoir-derived cell lines from bats could provide hints to a wildlife origin of human viruses that are already circulating in the human population for some time: For example, Huynh and coworkers showed replication of the human coronavirus HCoV-NL63 in immortalized lung cells from the North American tri-colored bat ( in bats .Rousettus aegyptiacus), indicating that this model is highly suitable to investigate the biology of filoviruses in cells derived from their presumed reservoir [A main application of reservoir-derived cell lines is the investigation of virus-host interactions upon controlled infection experiments. Bat cell lines have already served as a valuable tool to study virus entry and replication among a broad range of zoonotic viruses in models representing the natural reservoir host. For example, two filoviruses, Ebola virus (EBOV) and Marburg virus which are harbored by bats in the wild, were shown to enter and replicate efficiently in a bat cell line derived from the Egyptian fruit bat  may have a similar hantavirus entry receptor, although they are phylogenetically a long time separated as reflected in their morphological features [The above-mentioned differences in the host range of hantaviruses might be driven by polymorphisms in the receptor molecules. Thus, closely related features . In addifeatures . To studfeatures .It has been shown that hantaviruses can infect multiple cell types, but have a tropism to endothelial cells in human infection, a reason why human umbilical vein endothelial cells (HUVECs) are the main cell culture model for hantavirus disease in humans . Furthervs. spontaneously vs. artificially immortalized cells, see To ensure the continuous availability of a cell culture model system, immortalization is an option to create infinite cell lines from primary cells. While in primary rodent cells spontaneous immortalization can occur, it is not known if bat or insectivore cells behave in the same way. Other methods of immortalization include retroviral systems such as lentiviral transduction of the coding sequence of large T antigen of SV40 or introduction and stable expression of telomerase reverse transcriptase protein, both attempts which have been already successfully applied for the generation of bat cell lines ,81,102. To obtain suitable tissue of reservoir hosts, ongoing research projects on small mammals can be of use for collection of organ material to isolate primary cells. While it is not possible to generate cell lines from the plethora of rodent and insectivore species in which hantaviruses are found, a pragmatic approach is to focus on representative species. Selection criteria for these representative species could be: the overall importance of the associated virus, availability of breeding colonies, and representation of certain families/genera.in vivo infection in an animal model with the associated virus species. Further, laboratory-bred populations are less likely to be infected with unwanted pathogens, and in most instances, stem from a genetically characterized background. Natural reservoir host species that are available as laboratory animals, in breeding colonies or in enclosures  are of special interest: cell lines derived from these animals could be compared to the i.e., laboratory mice are an option. Thorough screening, for example by next generation sequencing of organ material, can provide further assessment to ensure high-quality cell lines without contaminants, even for as yet unknown pathogens. Furthermore, thorough characterization of the cell lines is obligatory for their use in virus infection studies. This characterization should focus on the following questions: Do these cells still express relevant receptors, are interferon signaling pathways still intact, do these cells still represent important characteristics of their cell type of origin . Although complex, this characterization ensures a valid cell culture model, which can then easily be shared between different research groups and lead to novel insights into the highly conserved hantavirus-host interaction in the context of their natural reservoir. As seen in the field of bat-borne zoonoses, reservoir-derived cell lines can serve as a valuable in vitro tool and therefore this approach should also be used in the field of rodent- and insectivore-borne zoonotic viruses. Furthermore, synergistic approaches of bat-, rodent- and insectivore-borne viruses and their reservoirs might enable the identification of general mechanisms of virus persistence, conserved across a broad range of mammalian reservoir hosts. Protocols for the generation of bat cell lines have been established and could be adapted to the rodent and insectivore host [If cell lines are generated from feral animals, a thorough screening for contaminants from the field is necessary . Animals from which tissue material was obtained should be either directly tested for contamination with known pathogens or alternatively sentinel animals, ore host ,81,102; In conclusion, investing in a large range of reservoir-derived cell culture models will be a promising tool to reveal novel aspects of the hantavirus-host relationship. Further, experience and model systems from the field of bat-borne zoonosis can serve as a blueprint for the hantavirus research community.


A 14-month-old boy presents for evaluation of fingers that are attached together as shown in the picture. The boy is diagnosed with syndactyly and prepared for surgery.What is syndactyly? How common is it?Discuss the embryologic basis, epidemiology, and environmental factors that can lead to syndactyly.How is syndactyly classified?What syndromes are most commonly associated with syndactyly?When is the optimal time to repair syndactyly?What are the basic principles of syndactyly repair?What are complications associated with syndactyly repair?Are multiple surgeries necessary for syndactyly repair?Syndactyly is a congenital hand anomaly defined by adjacent digits that fail to separate during embryological development. The most common congenital hand defect, syndactyly occurs at a rate of 1 in 2000 live births, with males twice as likely to be affected. Fifty-seven percent of cases present in the third web space, and 50% of cases are bilateral.Syndactyly occurs because of the fusion of soft tissue with or without bony fusion. The underlying defect results from the failure of interdigit mesenchymal tissue to undergo apoptosis during the seventh and eight week of gestation.2complete, while webbing that terminates more proximally is incomplete. A simple syndactyly refers to those with only soft tissue between digits, and a complex syndactyly refers to digits with bony or cartilaginous fusions. Complicated syndactyly refers to fingers with bony , musculotendinous, and/or neurovascular abnormalities.Although classified anatomically, embryologically, or genetically/molecularly, syndactyly is most commonly described by the extent of webbing, degree of fusion, and the presence of duplicated skeletal parts within the interdigital web space. If the webbing extends to the fingertips, it is considered to be -In patients lacking a positive family history, a higher incidence of syndactyly has been associated with maternal smoking, lower nutritional/economic status, and increased meat/egg consumption while pregnant.3-Syndactyly repairs are usually performed between 12 and 18 months of age to minimize scar contracture (operating too early) and deviation of the joints (operating too late). If syndactyly is complex or fingers are of significantly unequal size , correction is done at 6 months of age or even earlier5,5-5-5,9With numerous successfully documented methods for reconstruction, syndactyly repair has no gold standard operation. However, all methods employ a common set of techniques that any surgeon must be comfortable with before attempting a repair. Techniques include use of full-thickness flaps, triangular/zig-zag/rectangular incisions to separate digits, preservation of vascular supply to the digit, and a meticulous approach to reconstruction with attention to preserving anatomic proportions and details.5,9The most common acute postoperative complications of syndactyly repair are related to skin infection and necrosis, graft failure, and scar contracture related to a child's activity or lack of immobilization. These often require resurfacing with another graft and debridement. Long-term complications include web creeping, skin graft dysmaturation, keloid formation, and joint instability/deformity. “Web creeping” is the recurrence of abnormal webbing between digits that occurs because of scar contracture at the base of attachment or along incision lines.6Follow-up surgeries for syndactyly are usually not necessary. However, if severe contractures develop, a second graft or release will need to be done, and secondary operative rate has been reported to be around 10%.6


We compared microRNA expression patterns in three cisplatin resistant sublines derived from paternal cisplatin sensitive germ cell tumor cell lines in order to improve our understanding of the mechanisms of cisplatin resistance.Three cisplatin resistant sublines  showing 2.7-11.3-fold increase in drug resistance after intermittent exposure to increasing doses of cisplatin were compared to their parental counterparts, three well established relatively cisplatin sensitive germ cell tumor cell lines . Cells were cultured and total RNA was isolated from all 6 cell lines in three independent experiments. RNA was converted into cDNA and quantitative RT-PCR was run using 384 well low density arrays covering almost all (738) known microRNA species of human origin.Altogether 72 of 738 (9.8%) microRNAs appeared differentially expressed between sensitive and resistant cell line pairs  of which 46.7-95.3% were up-regulated . The number of genes showing differential expression in more than one of the cell line pairs was 34 between NTERA-2R/NTERA-2 (79%) and NCCIT-R/NCCIT (64%), and 3 and 4, respectively, between these two cell lines and 2102EP-R/2102EP (about 27%). Only the has-miR-10b involved in breast cancer invasion and metastasis and has-miR-512-3p appeared to be up-regulated (2-3-fold) in all three cell lines. The hsa-miR-371-373 cluster (counteracting cellular senescence and linked with differentiation potency), as well as hsa-miR-520c/-520h (inhibiting the tumor suppressor p21) were 3.9-16.3 fold up-regulated in two of the three cisplatin resistant cell lines. Several new micro-RNA species missing an annotation towards cisplatin resistance could be identified. These were hsa-miR-512-3p/-515/-517/-518/-525 (up to 8.1-fold up-regulated) and hsa-miR-99a/-100/-145 (up to 10-fold down-regulated).Examining almost all known human micro-RNA species confirmed the miR-371-373 cluster as a promising target for explaining cisplatin resistance, potentially by counteracting wild-type P53 induced senescence or linking it with the potency to differentiate. Moreover, we describe for the first time an association of the up-regulation of micro-RNA species such as hsa-miR-512-3p/-515/-517/-518/-525 and down-regulation of hsa-miR-99a/-100/-145 with a cisplatin resistant phenotype in human germ cell tumors. Further functional analyses are warranted to gain insight into their role in drug resistance. The paternal cell lines are (1) the p53-wt (wild type) pluripotent gonadal germ cell tumor cell line NTERA-2, (2) the p53-mutated pluripotent extragonadal nonseminomatous germ cell tumor cell line NCCIT and (3) the p53-wt nullipotent embryonal cell carcinoma 2102EP, derived from a primary human testicular teratocarcinoma. NCCIT cells have previously been shown to have an approximately 4-fold higher IC50 to cisplatin, compared to NTERA-2 [Cisplatin  is a chemotherapeutic agent widely used in the treatment of several solid tumors, among them testicular cancer, lung cancer, breast cancer, and bladder cancer. Resistance to cisplatin is a serious obstacle to effective cancer therapy. Clinically relevant levels of resistance can emerge quickly after treatment. Beside intrinsic resistance, acquired or gradually developing resistance has been observed in tumors under therapy. Several mechanisms underlying resistance have been described, like decreased exposure to the drug, e.g. via reduced drug accumulation/drug-target interaction or increased detoxification response, diminished cell-cycle effects, reduced apoptotic responses, or increased DNA-repair ,2. A num2 and were passaged every 3-4 days. After trypsination of cultured cells from steady state conditions and at comparable cell density, aliquots containing 10 × 106 cells were transferred into 1 ml RNA-later solution and stored at -20°C. Three aliquots per cell line originating from different cell passages  were processed and stored as described above.Both chemo sensitive paternal 2102EP  and NCCIT  cells as well as their cisplatin resistant sublines were cultured in DMEM F12 (1:1) medium containing 10% fetal calf serum . NTERA-2  and the cisplatin resistant subline were cultured in DMEM medium supplemented with Glutamax-I  containing 10% fetal calf serum . Cells were incubated at 37°C in a humidified atmosphere with 5% CO10 dose of each parental cell line. At reaching 50% cell kill, the addition of cisplatin was interrupted and cells were allowed to recover over three passages. Analyses presented here were performed after maintaining the resistant sublines for at least 1 week in cisplatin-free medium to allow for an adequate wash-out period. Results of cytotoxicity experiments of all six cell lines using the MTT assay -2,5-Diphenyltetrazolium Bromide) have been published [The cisplatin resistant sublines NTERA-2-R, 2102EP-R and NCCIT-R were generated by intermittent exposure of the parental cell lines to cisplatin over a time period of 18 month, starting with the respective ICCells were transported on dry ice in RNA-later solution  and stored at -80°C until use. After thawing the cells, they were disrupted using a denaturing lysis buffer, and RNA was isolated by combining organic extraction (phenol-chloroform) followed by immobilization of RNA on glass-fiber filters employing the mirVana miRNA Isolation Kit  in order to purify total RNA including small RNA species. Remaining DNA was digested on glass-fiber filters , and after performing quality controls, RNA was stored at -80°C until analysis by RTQ-PCR.To control the quality and purity (protein or DNA contamination) of isolated total RNA including small RNA, we performed spectral photometry, agarose gel electrophoresis, and PCR (using β-Actin primers). Only high quality total RNA including small RNA species was used for further experiments."Megaplex pools without preamplification protocol for microRNA expression analysis protocol". In a second step, the whole template cDNA and 450 μl 2x RT-PCR master mix were adjusted to a total volume of 900 μl by adding nuclease free water, and aliquots of 100 μl were pipetted into each fill port of a 384-well LDA. Cards were centrifuged twice , sealed, transferred into the 7900 RTQ-PCR instrument and a specific RTQ-PCR protocol was run over two hours using the 384-well LDA format. Two different LDAs existed, thus covering 738 human micro-RNAs. This made it necessary to create two kinds of cDNAs suitable for each of both LDAs using different sets of primers.Aliquots from each RNA sample (10 × 1 μg) were reversely transcribed without preamplification over three hours using the so called Normalization was performed using the median gene expression on each LDA separately, because this proved to be the more robust and slightly more precise method compared to a normalization approach using a housekeeping micro-RNA species provided on the LDA (data not shown). The median gene expression was subtracted from the CT-value of each of the spotted genes, following the ∆CT-quantitative approach for normalization purposes. Normalized gene expression results of cisplatin-resistant cell lines were expressed relative to the genes measured in the paternal chemosensitive cell lines by subtracting the corresponding CT-values (∆∆CT-quantitative approach). As a result, differential gene expression of cisplatin-resistant cell lines was expressed as a several-fold up- or down regulation of micro-RNAs relative to their paternal chemo sensitive origin. These ratios of corresponding cell line pairs are expressed such as e.g. NTERA-2-R/NTERA-2. Only ratios ≥2/≤0.5 were considered to represent differentially expressed genes. All the materials and instruments used for RTQ-PCR were ordered from Applied Biosystems, Weiterstadt, Germany.All technical procedures were performed in accordance to standard operating procedures (SOP) implemented in our laboratory in 2008 when the Institute became accredited according to DIN EN ISO 9001/2008.Statistical measures  were computed using SAS .50 to the substance, as measured by the MTT assay -2,5-Diphenyltetrazolium Bromide). Whereas the IC50 values of the sensitive parental cell lines were 0.45 μM , 0.75 μM (+/- 0.1), and 1.75 μM (+/- 0.1), respectively, the three cisplatin resistant sublines exhibited IC50 values of 5.1 μM (+/- 0.1) in NTERA-2R, 4.9 μM (+/- 0.1) in 2102EP-R, and 4.7 μM (+/- 0.2 ) in NCCIT-R. Results of the cytotoxicity experiments have been reported elsewhere [Intermittently culturing NTERA-2, 2102EP, and NCCIT cell lines exposed to increasing concentrations of cisplatin resulted in a 2.7 - 11.3 increase in ICThe coefficient of variation  was chosen to describe the observed methodological, intra- and interindividual variability of our three independently performed experiments. Mean CV values of differentially expressed genes and associated interquartile ranges appeared cell line dependent, showing increased mean CV values (25-75 percentile) starting at 25.3% (25-75 percentile: 15.7-29.0) in cell line pairs such as NCCIT-R/NCCIT, 30.5% (25-75 percentile: 18.5-36.3) in NTERA-2-R/NTERA-2, and 37.4% (25-75 percentile: 18.6-40.4) in 2102EP-R/2102EP cells . Altogether 72 of 738 genes appeared differentially expressed  appeared differentially expressed with considerable overlap in micro-RNA gene expression occurring between the cell line pairs NTERA-2-R/NTERA-2 and NCCIT-R/NCCIT, but considerably less overlap with the cell line pair 2102EP-R/2102EP cells  or about 10-fold down-regulated (hsa-miR-99a/-100/-145) in both NTERA-2-R/NTERA-2 and NCCIT-R/NCCIT cell line pairs. Literature results describing a role of these micro-RNAs in cisplatin resistance are missing, warranting further studies to elucidate their potential role in development of resistance.Recently, a role of the miR-106b seed family members in cisplatin resistance of testicular cancer has been described by another group . AlthougIn summary, our approach, simultaneously examining almost all known human micro-RNA species, confirms that the miR-371-373 cluster seems to be involved in cisplatin resistance in germ cell tumors in vitro and could be an interesting target for interference with drug resistance. Moreover, new micro-RNA species such as hsa-miR-512-3p/-515/-517/-518/-525 or hsa-miR-99a/-100/-145, also potentially involved in cisplatin resistance, could be identified in the germ cell tumor cell lines studied here. It will be of interest to examine tumor samples of patients with both cisplatin-sensitive and cisplatin-resistant germ cell tumors to analyse whether the changes described here are also found in vivo. Furthermore, functional analyses, e.g. by employing si-RNA and simultaneous whole genome microarrays, can help to examine the causal link with cisplatin resistance and to get insight into the mRNA species controlled by the micro-RNAs.The authors declare that they have no competing interests.MP designed the study, analyzed results and drafted the manuscript. SG carried out experiments related to induction of cisplatin resistance and drafted the manuscript. CR carried out RTQ-PCR experiments and performed statistical analysis. AR carried out RTQ-PCR experiments and analyzed the results. CB participated in the design of the study and coordination. VM participated in the design of the study and coordination. FH was responsible for the induction of cisplatin resistance experiments, designed the study and drafted the manuscript. MA designed the study, was responsible for RTQ-PCR experiments, analyzed results, performed statistical analysis and drafted the manuscript. All authors read and approved the final manuscript.Table S1: Complete list of all detectable miRNA examined. Differential gene expression relative to the corresponding gene of the paternal cell line - complete list of all detectable miRNAs examined (lying within the linear-dynamic range of our method)Click here for file


Cells were treated with cisplatin  and sunitinib malate , either in isolation or combined, over the course of 72 hours. 3--2,5-diphenyl tetrazolium bromide assay, acridine orange, and monodansylcadaverine staining and flow cytometry were performed. The combination index (CI) was calculated based on the Chou and Talalay method. In isolation, cisplatin and sunitinib malate statistically (P < 0.05) decrease cell viability in all cell lines in a dose-dependent manner, with the presence of autophagic vacuoles. A cell cycle arrest in early S-phase and in G0/G1-phase was also found after exposure to cisplatin and sunitinib malate, in isolation, respectively. Treatment of urinary bladder-cancer cells with a combination of cisplatin and sunitinib malate showed a synergistic effect (CI < 1). Autophagy and apoptosis studies showed a greater incidence when the combined treatment was put into use. This hints at the possibility of a new combined therapeutic approach. If confirmed in vivo, this conjugation may provide a means of new perspectives in muscle-invasive urinary bladder cancer treatment.The aim of this paper is to analyse sunitinib malate  Urinary bladder cancer is a common malignancy of the urinary tract, being four times higher in men than in women . RemarkaSunitinib malate is an orally bioavailable molecule with the ability to block the intracellular tyrosine kinase domain of tyrosine kinase receptors. It is also responsible for inhibition of vascular endothelial growth factors receptors, plateletderived growth factor receptors, and stem cell factor receptor , 9. Its in vitro effects of cisplatin and sunitinib malate in isolation and in combination, on one human nonmuscle invasive urinary bladder-cancer cell line (5637) and on two human muscle-invasive urinary bladder-cancer cell lines (T24 and HT1376).This investigation aims to analyse the μg/mL streptomycin , 100 U/mL penicillin , and 2 mM L-Glutamine . The cultures were maintained in a humidified 5% CO2 incubator at 37°C.The study was performed on the 5637, T24, and HT1376 urinary bladder-cancer cell lines. T24 cell line was provided by DSMZ, Düsseldorf, Germany; 5637 and HT1376 cell lines were kindly provided by Dr. Paula Videira of the Universidade Nova de Lisboa, Lisboa, Portugal. Monolayer cultures of the three cell lines were maintained in RPMI 1640 medium , supplemented with 10% heat inactivated fetal bovine serum , 100 μM cisplatin  and 1, 2, 4, 6, and 20 μM sunitinib malate , in isolation and over the course of 72 hours to assess dose-response profiles. For the cell viability combined assay, 3, 6, and 13 μM cisplatin was used with each concentration of sunitinib malate . For morphological analysis, acridine orange, and monodansylcadaverine (MDC) staining, the lowest concentration of cisplatin (3 μM) was used simultaneously with 1 μM of sunitinib malate. For flow cytometry assay, 3 μM cisplatin was combined with 1, 2, and 4 μM of sunitinib malate.T24, 5637, and HT1376 urinary bladder-cancer cells were exposed to 3, 6, 13, and 18 Cells growing in the complete medium alone were processed in the same way as the treated cells, in the case of all the methodologies, and cytotoxic effects were analyzed immediately after drug exposure was ceased.4 cells/mL) were cultured in the presence of the lowest concentrations of cisplatin and sunitinib malate, in isolation or combined. A light inverted microscope  was used to observe the cells in culture, in order to detect cells confluence and differences in their appearance.Urinary bladder-cancer cell lines  and sunitinib malate , in isolation or combined. At the end of the treatment, the MTT  dye working solution (10 μL/well at 5 mg/mL) was added to each well and plates were incubated for 4 additional hours. The medium was removed and the formazan crystals generated were solubilized by adding 100 μL/well of dimethylsulfoxide (Sigma Aldrich) for 5 minutes. Absorbance values at 492 nm were determined using an automatic ELISA plate reader . The percentage of cell viability was calculated as (absorbance of treated cells/absorbance of untreated cells) ×100.The effect of both drugs was evaluated based on the MTT assay. Cells were seeded into each well of a 96-well flat-bottom microtiter  at a density of 2 × 101/(Dx)1 + (D)2/(Dx)2 + (D)1(D)2/(Dx)1(Dx)2, where (D)1 and (D)2 are the concentrations of sunitinib malate and meloxicam that exhibit a determined effect when applied simultaneously to the cells and (Dx)1 and (Dx)2 are the concentrations of the same drugs that exhibit the same determined effect when used in isolation. The CI values indicate a synergistic effect when <1, an antagonistic effect when >1, and an additive effect when equal to 1.For the study of synergism between cisplatin and sunitinib malate on cell growth inhibition of T24, 5637, and HT1376 cells, a combination index (CI) was performed using the data obtained from MTT assay. Drug combination studies were based on concentration-effect curves generated as a plot of the fraction of unaffected cells versus drug concentration, in accordance to the Chou and Talalay (1984) method , using t4 cells/mL) were seeded on sterilized glass coverslip (8 mm), cultured for 24 hours, and treated with drugs, in isolation or combined, for 72 hours. The medium was removed and acridine orange  was added to the cells at 1 μg/mL, at 37°C and for 10 minutes. Subsequently, cells were washed with phosphate-buffered saline (PBS) and immediately analysed using a fluorescence microscope .Acridine orange is a fluorescent dye which stains cytoplasm and nucleus by bright green, while acidic compartments (such as lysosomes and autolysosomes) stain bright red. Cells (2 × 104 cells/mL) were seeded in sterile coverslips (8 mm), incubated for 24 hours, and then treated with the both drugs, as single agents or in combination, for 72 hours. Autophagic vacuoles were labelled with MDC by incubating cells with 25 μM MDC for 1 hour at 37°C. The cells were washed three times with PBS and immediately analysed using a fluorescence microscope .Autophagy induced by cisplatin and sunitinib malate, in isolation or combined, was observed with the autofluorescent substance MDC . MDC moves freely to cross-biological membranes and accumulates in autophagic vacuoles . Cells (6 cells/mL) were seeded in 6-well plates and allowed to adhere overnight. Subsequently, the medium was removed and 3 μM cisplatin, in isolation or in combination with sunitinib malate  was applied. Cell-cycle distribution and apoptosis were analyzed by flow cytometry as previously reported [0/G1 phase cells were considered to be apoptotic (sub-G0/G1) [Cells (1 × 10reported . DNA-conb-G0/G1) . Each inF test and the statistical significance of differences between the treatment and control groups was determined by Dunnett's multiple comparison post hoc test for the MTT assay. The Pearson product-moment correlation coefficient was used to evaluate the correlation (linear dependence) of the cell-cycle and drugs concentration. Data obtained from MTT assay and used to evaluate the interaction between cisplatin and sunitinib malate were analyzed using the MATLAB software . Statistical significance was set at P < 0.05.Statistical analysis was carried out using the SPSS 17.0 statistical software . The equality of variances was tested by the Levene Cisplatin and sunitinib malate, in isolation, induced a decrease cell population when compared to untreated cells. In combined treatment, a slight decrease of cell confluence with an increase number of granulated cells was observed, when compared with the other culture flasks with isolated drugs. These features were more visible on T24 and 5637 cell lines. The surface in control flask was confluent with visible undergoing division cells .T24, 5637, and HT1376 cell lines in the exponential growth phases were exposed to different concentrations of cisplatin and sunitinib malate, in isolation or combined, and the effect on cell viability was examined after 72 hours of culture.μM). In the three cell lines, statistically significances (P < 0.05) were found in all the concentrations tested when compared with untreated cells . In all the cell lines, statistically significant values were found when compared with untreated cells (P < 0.05), with the exception at the lowest concentration in the HT1376 cell line (P = 0.171)  .μM) and sunitinib malate  decreased the cell viability rate in the three cell lines when compared with each drug in isolation (μM) with 1, 2, and 4 μM concentration of sunitinib malate. The 5637 survival rates for this combination were averaged as 53.2%, 33.1%, and 29.7%, followed by the T24  and HT1376  cell lines, respectively. All the combinations are statistically significant when compared with the control group (P < 0.05).The simultaneous treatment of urinary bladder-cancer cells to cisplatin  represents the magnitude of dose reduction obtained for the 50% growth inhibitory effect in combination setting as compared to each drug alone. In our experiments, DRI50 of cisplatin and sunitinib malate were equal to 5.51 and 1.5 in HT1376, 2.86, and 2.19 in T24, and 1.25 and 20 in 5637 cells, respectively, when the two drugs were used in combination  between the cisplatin and sunitinib malate in combination at 72 hours on T24, 5637, and HT1376 cell lines, we implemented on MATLAB the method developed by Chou and Talalay (1984) . The CI5ectively . Therefobination . These rμM) and sunitinib malate (1 μM). However, this effect was more evident on sunitinib malate treated cells. Upon exposure to combined treatment, increased acidic vesicular organelles was detected in the three cell lines. Moreover, with this staining it was possible to observe that cells exposed to cisplatin, in isolation, showed the formation of membrane blebbing, which are morphological alterations consistent with apoptosis. This detection had a higher incidence on T24 and 5637 cell lines and markedly increased in the combined treatment  was observed in cytoplasm and perinuclear regions .0/G1-phase, with 75.9%, 91.6%, and 81.1%, on HT1376, T24, and 5637 cell lines, respectively. With 3 μM cisplatin the percentage of cells in G0/G1-phase decreased to 45.9% (HT1376), 71.5% (T24), and 24.1% (5637). This decrease was accompanied by an early S-phase and sub-G0/G1 arrest, particularly for 5637 and T24 cell lines. Regarding sunitinib malate treatment, two cell cycle effects were observed for all the cell lines. First the cells in interphase were mainly in G0/G1-phase  and ), respectively,  and two muscle-invasive cell lines (T24 and HT1376).Urinary bladder cancer is a common malignancy and remains a challenge despite significant therapeutic advances . Thus, nP < 0.05) reduced cell viability in a dose-dependent manner as already reported in our previous studies [in vivo studies, sunitinib malate was effective in mouse with small cell lung cancer [The treatment of HT1376, T24, and 5637 urinary bladder-cancer cell lines with cisplatin and sunitinib malate, in isolation, significantly . Sonpavde and collaborators (2009)  have alrl effect .The different response obtained on the three cell lines, being the nonmuscle invasive urinary bladder-cancer cell line the most sensitive, may be explained by the different origin of the cell lines. Nevertheless, the two muscle-invasive urinary bladder-cancer cell lines presented a similar and improved pattern response, when compared with each drug in isolation.in vitro and in vivo studies are required to confirm these results. The present data opens a new possible approach in the treatment of muscle-invasive urinary bladder cancer.In conclusion, and although this is a preliminary study, this is the first report that provides valid results concerning the combination of cisplatin and sunitinib malate, on muscle-invasive urinary bladder-cancer cell lines. This synergistic interaction leads to a reduced cell viability, increased autophagy, and apoptosis. Future molecular


The complete list of 115 proteins identified as being hit uniquely by compounds showing selective antiproliferative effects for tumor cell lines is provided.A novel chemocentric approach to identifying cancer-relevant targets is introduced. Starting with a large chemical collection, the strategy uses the list of small molecule hits arising from a differential cytotoxicity screening on tumor HCT116 and normal MRC-5 cell lines to identify proteins associated with cancer emerging from a differential virtual target profiling of the most selective compounds detected in both cell lines. It is shown that this smart combination of differential  Cancer is a disease of the cell The advent of high-throughput cell-based cytotoxicity assays opened new perspectives for anticancer discovery In recent years, the availability of an increasing amount of protein-ligand interaction data in the public domain has promoted the development of ligand-based computational methods aiming at predicting the affinity profile of small molecules across multiple targets The integration of differential cytotoxicity screening and virtual target profiling for the identification of cancer-relevant targets was put into practice within the context of CancerGrid, a European Commission project under Framework Programme 6 in vitro and in silico screening (DIVISS) process followed in this work is depicted in For the sake of clarity, a summary scheme of the overall differential A cell-based cytotoxicity screening campaign was performed on a chemical collection composed of 30,000 diverse molecules selected mainly from the entire AMRI catalogue 50 values obtained in MRC-5 cells, IC50 (MRC-5), and those obtained in HCT116 cells, IC50 (HCT116), was derived for each compound. A total of 230 compounds were identified to be 5 times or more cytotoxic in tumor cells than in healthy cells (IC50 MRC-5/IC50 HCT116≥5). A chemotype clustering analysis in vitro screening, leading to the identification of 265 compounds with selective cytotoxicity for tumor cells  and 17--17-demethoxygeldanamycin (17AAG) were selected as representative pan-HDAC and HSP90 inhibitors, respectively. Dose-response curves on both HCT116 and MRC-5 cell lines were determined for the two inhibitors . The res50 of 80 nM for MAO-B, in good agreement with our predictions. In addition, it was also tested at multiple human tumor cell lines, including six colon cancer cell lines. Among them, the pGI50 value reported in PubChem for colon HCT116 cell lines (4.64) is, within the variability limits of this type of experiments, in good agreement with the pGI50 value obtained in this work for the same type of cell lines (5.19). The dose-response curve of the cytotoxicity of NSC680350 on HCT116 cell lines in this work and a summary of all colon cancer data found in PubChem for this compound is provided in We checked also whether within the set of 265 compounds showing selective antiproliferative effects for tumor cell lines there was any compound that could have been tested on a range of colon cancer cell lines and for which screening data was also available in the public domain. Much to our surprise, we found experimental data in PubChem Substantiation of the potential relevance to cancer of the list of 115 proteins identified as being targeted solely by tumor selective compounds was performed by two independent perspectives. On the one hand, all 115 proteins were scored on the basis of recently derived oncogene probabilities (OncoScores) and checked for currently available experimental data on the up- and down-regulation in colon cancer samples The OncoScores for all 115 proteins targeted by tumor selective compounds were obtained from the CGPrio website The subset of 42 tumor selective proteins with OncoScore higher than 0.7 is provided in 50 value of 1.62 µM A close look at the top-20 ranked proteins present in bona fide cancer targets, such as HSP90, with a low CGPrio probability. In this respect, the low OncoScore obtained for HSP90 means only that, on the basis of current knowledge on cancer genes, HSP90 does not share properties with the rest of cancer genes for which information is available. Taken together, these results emphasize the potential applicability of the DIVISS approach as a complementary strategy to the identification of cancer-relevant targets.It may also surprise that currently recognised cancer targets, such as HSP90, are not present in The BioCarta resource in vitro data on the interaction of drugs with multiple proteins has increased dramatically In recent years, the amount of publicly available in silico target profiling, will be selectively associated to each cell line.There are two recognisable extensions to the version of the DIVISS approach presented here. The first obvious extension is in the use of other cell lines. In this particular study, HCT116 and MRC-5 cell lines have been taken as models of tumor and healthy cell lines, respectively. However, there are numerous alternative human tumor cell lines that can be used instead and those can in turn be differentially compared to several healthy cell lines as well in vivo efficacy through modulation of multiple targets rather than selective interaction on a single target. The big question is then defining the essential protein signature of each cancer type, so it can be thoroughly addressed by novel cancer therapeutic agents Cell systems are implicitly robust and selectively acting on one particular target may not be the most efficacious way of modulating or interfering with them as the system may always find ways to compensate for the selective perturbation incorporated. Instead, targeting multiple essential targets in tumor cells may be a more efficient strategy to make more difficult for the cell system to compensate for all perturbations introduced. Indeed, recent evidences indicate that most cancer drugs attain their The CancerGrid consortium had privileged access to the entire chemical catalogue at AMRI 2 incubator. Subculturing was done using 1∶5 ratio twice a week. MRC-5 cells were maintained in DMEM supplemented with 10% FBS, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified 5% CO2 incubator. Subculturing was done using 1∶3 ratio twice a week. A cell bank was established for the cell lines used during the differential toxicity screening campaign. In all experiments, the doubling time between cancer and normal cells was approximately 24 hours.A colon adenocarcinoma (HCT116) and normal human lung fibroblast (MRC-5) cell lines were purchased from the American Type Culture Collection . HCT116 cells were maintained in McCoy's 5a Modified Medium supplemented with 10% FBS, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified 5% COin vitro assay for measuring toxicity and antiproliferative effects of small molecules was implemented as described earlier 2 incubators at 37°C for 48 h. This incubation period has the advantage of allowing those compounds that are not directly toxic but can block or slow down cell proliferation to have enough time to show their effect. During this period, FBS content of the medium was decreased to avoid the masking effect of FBS on toxicity. Detection of viability is based on the reduction of resazurin (Alamar blue) by living cells, resulting in an increased fluorescent signal 50 values were determined and analyzed. To test the reproducibility and robustness of the assay for high-throughput screening, Z′ factors (∼0.72) and S/B ratios (∼10) were determined and the respective plate-to-plate and day-to-day coefficients of variation found to be 5.0%, 2.7%, 1.7% and 5.6%. Based on the established assay protocol, single-point screenings were done at 50 µM compound concentration in duplicate. Likewise, IC50 values were obtained from the toxicity dose-response curves from six compound concentrations in duplicate and calculated with Microcal Origin 5.0. Compounds showing selective cytotoxicity for tumor cells relative to healthy cells are identified by large values of the ratio IC50(MRC-5)/IC50(HCT116), whereas the inverse of this ratio serves to recognize compounds with selective cytotoxicity for healthy cells relative to tumor cells.The high-throughput There are currently several public sources that contain chemical structures with information on the binding or functional activity to protein targets. Those used in the present work include ChEMBLdb To be processed efficiently, molecular structure information needs to be encoded using some sort of mathematical descriptors. In this work, three types of two-dimensional descriptors were used, namely, SHED, FPD, and PHRAG Expression profiles for proteins in various types of cancer were directly extracted from IntOGen SAHA, a pan-HDAC inhibitor, was kindly provided by Ciro Mercurio  with 95% purity and 17AAG, a HSP90 inhibitor, was obtained from Sigma  with 95% purity.Figure S1Distributions of pairwise similarities using PHRAGS (top) and PFPD (bottom) descriptors between compounds with selective cytotoxicity in HCT116 cell lines (left), MRC-5 cell lines (middle), and HCT116 and MRC-5 cell lines (right).(PDF)Click here for additional data file.Figure S2List of chemical structures showing selective cytotoxicity for HCT116 cell lines.(PDF)Click here for additional data file.Figure S3List of chemical structures showing selective cytotoxicity for MRC-5 cell lines.(PDF)Click here for additional data file.Figure S4Dose-response curves of 17AAG, an HSP90 inhibitor (left), and SAHA, a HDAC inhibitor (right), on the HCT116 and MRC-5 cell lines.(PDF)Click here for additional data file.Figure S5Dose-response curve (left) of the cytotoxicity of compound NSC680350  on HCT116 cell lines (GI50 = 6.4 µM). Also provided (right) are the pGI50 values of the compound on the two cell lines tested in this work and the six colon cancer cell lines for which data is available in PubChem.(PDF)Click here for additional data file.Table S1List of all 115 proteins identified by the DIVISS approach as from small molecule hits selective to HCT116 relative to MRC-5. The OncoScore is the oncogene probability calculated with CGPrio (DOC)Click here for additional data file.Table S2List of all interactions available in PubChem for compounds present within the list of 265 cytotoxic selective in HCT116 cell lines.(DOC)Click here for additional data file.


Improving the treatment of renal cell carcinoma (RCC) will depend on the development of better biomarkers for predicting disease progression and aiding the design of appropriate therapies. One such marker may be fatty acid binding protein 7 (FABP7), also known as B-FABP and BLBP, which is expressed normally in radial glial cells of the developing central nervous system and cells of the mammary gland. Melanomas, glioblastomas, and several types of carcinomas, including RCC, overexpress FABP7. The abundant expression of FABP7 in primary RCCs compared to certain RCC-derived cell lines may allow the definition of the molecular components of FABP7's regulatory system.FABP7 mRNA levels in six RCC cell lines. Two were highly expressed, whereas the other and the embryonic kidney cell line (HEK293) were weakly expressed FABP7 transcripts. Western blot analysis of the cell lines detected strong FABP7 expression only in one RCC cell line. Promoter activity in the RCC cell lines was 3- to 21-fold higher than that of HEK293. Deletion analysis demonstrated that three FABP7 promoter regions contributed to upregulated expression in RCC cell lines, but not in the HEK293 cell. Competition analysis of gel shifts indicated that OCT1, OCT6, and nuclear factor I (NFI) bound to the FABP7 promoter region. Supershift experiments indicated that BRN2 (POU3F2) and NFI bound to the FABP7 promoter region as well. There was an inverse correlation between FABP7 promoter activity and BRN2 mRNA expression. The FABP7-positive cell line's NFI-DNA complex migrated faster than in other cell lines. Levels of NFIA mRNA were higher in the HEK293 cell line than in any of the six RCC cell lines. In contrast, NFIC mRNA expression was lower in the HEK293 cell line than in the six RCC cell lines.We determined FABP7 promoter regions drive reporter gene expression in RCC cell lines, but not in the HEK293 cell line. BRN2 and NFI may be key factors regulating the expression of FABP7 in certain RCC-derived cell lines.Three putative  Among primary renal tumors the most common is renal cell carcinoma (RCC). Although earlier detection of RCC has positively influenced patient outcomes , predictFABP7 belongs to a mammalian family of at least nine proteins that are specifically expressed in diverse tissues such as liver, intestine, heart, adipose tissue, epidermis, brain, peripheral nervous system, and testis . SeveralFABP7 transcripts in tumors or urine of patients with RCC  dATP, Perkin Elmer Inc. ; T4 Polynucleotide Kinase, Toyobo ; KCl, glycerol and NP-40, Wako .Reagents and their sources were the following: GIBCO® RPMI Media 1640 supplemented with 10% (v/v) fetal bovine serum . Cells were cultured at 37°C in a humidified atmosphere containing 5% (v/v) CO2.RCC cell lines  were purchased from the American Type Culture Collection . Caki-1, HEK293, OS-RC-2, and TUHR14TKB were provided by the RIKEN . All cell lines were grown in GIBCO® III Reverse Transcriptase primed by 500 ng of oligo (dT)12-18 according to the manufacturer's recommendations. PCR amplification was performed using 1 μL of first-strand complementary DNA (cDNA) as template with the primers and number of cycles listed in Table FABP7 and BRN2 expression in RCC and HEK293 cell lines were done with the Applied Biosystems StepOnePlus™ (Life Technologies). The final PCR reaction mix included 2 μL of 5 μM specific primer, 1 μL first-strand cDNA and 10 μL SYBR® Green PCR Master Mix at a final volume of 20 μL. Specific plasmid controls for FABP7, BRN2 and TBP were synthesized as the direct insertion of PCR products into a pCR®2.1-TOPO® vector, and standard curves for each marker were generated with seven serial dilutions of plasmid templates (0.1 nM to 0.1 fM). TBP was used as an internal control. The sense primer for FABP7 amplification, primers for TBP amplification and primers for OCT6 amplification were reported by Teratani et al.  dATP using T4 polynucleotide kinase and then purified using Illustra MicroSpin G-25 columns. Nuclear extracts were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents. For each assay, 5 μg of nuclear extract was used. Binding reactions were performed in 30 μl binding buffer  glycerol, 0.1% (v/v) NP-40, and 2 μg of poly(dI-dC)). Two microliters of 5 μM unlabeled competitor oligonucleotide or 1 μL of 2 μg/μL antibody were added as appropriate. Following addition of the nuclear extract and incubation for 30 min on ice, 1 μL of 100 nM labeled probe was added and incubated for 20 min at room temperature. DNA binding reactions were electrophoresed through a 6% (w/v) polyacrylamide gel in 0.5 × TBE to separate unbound probe from probe-protein complexes.Gel retardation assays were carried out using a slightly modified version of the method described by Bisgrove et al. . ComplemThe BRN-2 siRNA sequences used were as follows: 5'-GTGCAGACGCCCGTCCAG-3'  and 5'-cDNA: complementary DNA; FABP7: fatty acid binding protein 7; NFI: nuclear factor I; miRNA: microRNA; mRNA: messenger RNA; Q-PCR: quantitative polymerase chain reaction ; PCR: polymerase chain reaction; RCC: renal cell carcinoma; RT-PCR: reverse transcriptase-polymerase chain reaction; siRNA: small interfering RNA; SDS: sodium dodecyl sulfate; TBP: TATA box binding protein; YY1: Yin Yang 1.The authors declare that they have no competing interests.NT designed the study, performed the experiments, analyzed the data, and wrote the manuscript. TT1 participated in designing the study, analyzing the data and critically editing the manuscript. TT2, TS, SM and SO participated in critically editing the manuscript. All authors read and approved the final version of the manuscript.Data of Q-PCR and luciferase reporter assay. Figure Click here for fileFABP7 Western blot covering the full range. Western blot was performed using cytoplasmic extracts from HEK293 and RCC  cell lines.Click here for fileGel shift analysis with the DP probe. A) 32P-labeled DP (Table 32P-labeled oligonucleotide probes.DP Table  was addeDP Table . A 100-fClick here for file