Nature, the Cancer Cell Line Encyclopedia and the Cancer Genome Project, provide a wealth of data regarding the drug sensitivity and genomic profiles of many different types of cancer cells. In the present study we have mined these two studies for new therapeutic agents for SCLC and identified heat shock proteins, cyclin-dependent kinases and polo-like kinases (PLK) as attractive molecular targets with little current clinical trial activity in SCLC. Remarkably, our analyses demonstrated that most SCLC cell lines clustered into a single, predominant subgroup by either gene expression or CNV analyses, leading us to take a pharmacogenomic approach to identify subgroups of drug-sensitive SCLC cells. Using PLK inhibitors as an example, we identified and validated a gene signature for drug sensitivity in SCLC cell lines. This gene signature could distinguish subpopulations among human SCLC tumors, suggesting its potential clinical utility. Finally, circos plots were constructed to yield a comprehensive view of how transcriptional, copy number and mutational elements affect PLK sensitivity in SCLC cell lines. Taken together, this study outlines an approach to predict drug sensitivity in SCLC to novel targeted therapeutics.There are currently no molecular targeted approaches to treat small-cell lung cancer (SCLC) similar to those used successfully against non-small-cell lung cancer. This failure is attributable to our inability to identify clinically-relevant subtypes of this disease. Thus, a more systematic approach to drug discovery for SCLC is needed. In this regard, two comprehensive studies recently published in  Small cell lung cancer (SCLC) represents 15% of all lung carcinomas and is typically diagnosed when the disease has metastasized Nature, the Cancer Cell Line Encyclopedia (CCLE) A different approach to drug discovery for SCLC is needed and may lie in mining available databases on the drug sensitivities of SCLC cell lines. That is, as most SCLC cells are derived from metastatic sites or pleural effusions, they may be representative of extensive disease SCLC and its associated drug vulnerabilities. In this regard, two comprehensive drug-screening studies recently published in In the present study we have specifically extracted the data on SCLC cell lines from these two studies and outline a bioinformatic approach to identify new therapeutics for SCLC using polo-like kinase (PLK) inhibitors as an example.50≤1 µM), represented by paclitaxel. ‘Ineffective’ drugs, represented by erlotinib and sunitinib, produced no growth inhibition in most SCLC cells (IC50≥8 µM), although ‘outliers’ may be present. ‘Selective’ drugs, represented by rapamycin, demonstrated a long boxplot and can be considered effective for only a subset of SCLC cell lines.Initially we sought a global view of SCLC drug sensitivity in the CCLE 50 just outside 1 µM include AZD-7762, a CHK inhibitor; and JW-7-52-1, an MTOR inhibitor. HSP90 (17-AAG) and HDAC (panobinostat) inhibitors may also represent ‘effective’ drugs, although their efficacy varied among the two studies. ‘Effective’ drugs likely carry the best translational potential.As shown in Gene array data has been used extensively in cancer research to identify expression ‘signatures’ that may be either prognostic or predictive of tumor behavior. Therefore, we examined if gene expression clustering could be used to identify subgroups of drug-sensitive SCLC cell lines, particularly for drugs that demonstrated a broad efficacy range against SCLC cells in We next determined if CNV clustering could be used to identify subgroups of drug-sensitive SCLC cell lines. Three clusters were again identified using all 426 interrogated genes ; however50 values for the PLK inhibitors in most cell lines is between 10-100 nM, supporting our hypothesis that SCLC cells are broadly sensitive to PLK inhibitors.Our analyses identified PLK inhibition to have promising efficacy and little clinical trial activity in SCLC. Therefore, we used PLK inhibition as an example of how to use the CGP datasets to develop a genomic profile of SCLC drug sensitivity. First we sought to validate the efficacy of PLK inhibitors in SCLC. In these validation experiments we used SCLC cell lines, as well as inhibitors, that were different from those used in the original studies to highlight the efficacy of these new therapeutic agents. The SCLC cell lines used were H1048, H1688, SW1271 and DMS454. PLK inhibitors included BI-6727 (volasertib) and ON-01910 (rigosertib). In these experiments irinotecan served as a positive control while erlotinib served as a negative control. The results are shown in 50 values. We identified a list of 185 genes that were significantly differentially expressed between these two groups  with that for the five least sensitive SCLC cell lines ; as defined in the CGP by their BI-2536 ICTo validate that the PLK gene signature does, in fact, predict sensitivity to PLK inhibitors, we determined the efficacy of the PLK inhibitor BI-6727 in a cell line, H1092, which had gene expression data but no PLK sensitivity data in the CGP study, represented by a grey box at the top of It was of interest to determine whether the PLK gene signature was present in patient tumors and could potentially be used to predict tumor sensitivity to PLK inhibitors. The largest study of SCLC tumor gene expression is that of Rudin et al. TP53 or RB1, and sometimes both genes, whereas all sensitive cells displayed mutations of unknown protein significance (intronic and missense), typically in only one of these genes. All but one of the gene mutations was homozygous, indicating that resistant cells likely have no functional RB1 or TP53 protein. All sensitive cells display MYC , MYCN  or MYCL (CORL88) amplification, whereas only one resistant cell line displays MYC amplification (H2171). There also seems to be little or no CNV on the X chromosome in sensitive cells relative to resistant cells, which typically display some CNV loss. Genes on chromosome 13 are generally upregulated in PLK sensitive cells and downregulated in PLK resistant cells, whereas genes located on chromosome 19 are generally downregulated in PLK sensitive cells and upregulated in PLK resistant cells. Taken together, these results suggest that gene expression, CNV and mutational status may all contribute to the sensitivity of SCLC cells to PLK inhibition.Finally, we used circos plots Small-cell lung cancer is a disease in urgent need of new drug therapies. Unfortunately, the limited availability of tumor tissue hinders the acquisition of complete genomic analyses required for the identification and validation of new drugable targets in this cancer. Thus, alternative drug discovery strategies need to be developed until our understanding of the genomics driving SCLC can begin to approximate that of NSCLC, which benefits from comprehensive analyses of large cohorts of patient tumors, such as the The Cancer Genome Atlas (TCGA).In this report we have taken a bioinformatic approach to drug discovery for SCLC by data mining two large drug screening studies in cultured cell lines, the CCLE The CGP study also identified drug sensitivities that tended to cluster across all cell lines  and 53 SCLC cell lines examined. None of these PLK mutated cell lines were included in our study. Our PLK gene signature does, however, include genes on chromosomes typically deleted  and amplified (19p) in SCLC It was recently reported that mutations in the MYC-amplified cell lines, they also found the PLK inhibitor BI-2536 to be active, similar to our results, but did not pursue it further. They also showed that the majority of cells sensitive to the Aurora kinase inhibitor VX-680 demonstrated MYC-amplification. Interestingly, the CGP also included two Aurora kinase inhibitors (VX-680 and ZM-447439) in their study; however, it did not find any significant clustering of PLK with Aurora kinase inhibitors, indicating no link between these two classes of inhibitors when analyzed in the general population of cancer cell lines.During our study a report by Sos et al. In the present study we consistently identified three subtypes of SCLC cell lines using unsupervised clustering of either gene expression or CNV datasets from the CGP or CCLE  studies.Other unique approaches have been taken to identify new and effective therapies for SCLC. Jahchan et al. identified a surprising sensitivity of SCLC cells to tricyclic antidepressants in a drug repositioning study, which used bioinformatics to find drugs that induced changes in gene expression opposite to the gene expression profile of SCLC cells All cells were obtained from the ATCC and grown in their recommended medium. Cell proliferation was determined quantitatively by fluorescent DNA assay 50), gene expression, CNV, and mutation data was downloaded from http://broadinstitute.org/ccle (CCLE) and http://cancerrxgene.org (CGP) http://www.r-project.org/).The publically available CCLE and CGP drug sensitivity (IC50 greater than 8 µM in the CGP were thresholded at 8 µM. Boxplots were drawn for the IC50 from the two datasets respectively using R (http://www.r-project.org/). Mosaic plots were drawn using proc sgrender in SAS version 9.3 .For SCLC only, 24 drugs and 53 cell lines were included in the CCLE, while 92 drugs and 31 cell lines were included in the CGP. All ICNormalization of gene expression data  involved four steps: 1) raw data were normalized via the robust multi-array average (RMA) method; 2) probes without a gene name were removed; 3) gene level data was obtained by averaging the probe value within each gene and 4) the gene level data was then standard normalized by gene. For the gene expression data, the CCLE included 51 cell lines, while the CGP included 30 cell lines, among which three cell lines had duplicate measurements. Averaging the duplicates generated 27 unique cell lines for the CGP. Unsupervised consensus clustering was performed on the normalized gene level data with the R package “Consensus Cluster Plus”; parameters were set as default except maxK was set at 10. The Kruskal-Wallis test was used to assess expression differences between subtypes based on the consensus clustering results. Unsupervised hierarchical clustering was then performed using the significant genes only  and visualized using heatmaps via the R package “gplots”.Copy number data was obtained for the CGP data only for 426 genes The fastq data was aligned with tophat 2.0.9 Figure S1Boxplot of drug sensitivity in SCLC cells using the CCLE dataset. There are 53 cell lines for small cell lung cancer. The boxplots show drugs listed on the x-axis and the corresponding IC50 values (in µM) listed on the y-axis, similar to (PDF)Click here for additional data file.Figure S2Mosaic plot of drug sensitivity using CNV clustering of SCLC cells. Drug sensitivity was color-coded according to the legend at the bottom. Drugs are grouped along the y-axis according to their target molecule. Cells are arranged along the x-axis identical to their gene expression clustering identified in (TIF)Click here for additional data file.Figure S3Full-size circos plots of SCLC cells. Circos plots are shown full-size as individual panels for all the SCLC cell lines shown in collectively (PPTX)Click here for additional data file.Figure S4Unsupervised clustering of SCLC cells by gene expression using the CCLE dataset. Unsupervised consensus clustering was performed using the all 53 cell lines (only 51 gene expression available) and showed that 3 clusters was optimal for this dataset. With this assignment, we performed non-parametric one way ANOVA  test on those 3 clusters and obtained 4749 significant genes. We then generated the heatmap with those significant genes.(TIF)Click here for additional data file.Table S1Numerical data for drug efficacy determined in the CGP study. The 25%, 50% and 75% quantiles for all 92 drugs used to construct the boxplot in (DOC)Click here for additional data file.Table S2Numerical data for drug efficacy determined in the CCLE study. The 25%, 50% and 75% quantiles for all 24 drugs used to construct the boxplot in (DOC)Click here for additional data file.Table S3Significant genes used in CGP and CCLE gene expression clustering. The 1006 and 4749 significant genes used to cluster SCLC cell lines in the CGP ( the CGP  and CCLE the CGP  datasets(XLS)Click here for additional data file.Table S4Significant genes used in PLK gene expression clustering. The 185 significant genes used to cluster SCLC cell lines based upon their sensitivity to BI-2536 ( BI-2536  using th(XLS)Click here for additional data file.


Although advanced esophageal squamous-cell carcinoma (ESCC) is treated using a multidisciplinary approach, outcomes remain unsatisfactory. The microenvironment of cancer cells has recently been shown to strongly influence the biologic properties of malignancies. We explored the effect of supernatant from esophageal fibroblasts on the cell growth and chemo-resistance of ESCC cell lines.We used 22 ESCC cell lines, isolated primary human esophageal fibroblasts and immortalized fibroblasts. We first examined cell proliferation induced by fibroblast supernatant. The effect of supernatant was evaluated to determine whether paracrine signaling induced by fibroblasts can influence the proliferation of cancer cells. Next, we examined the effects of adding growth factors HGF, FGF1, FGF7, and FGF10, to the culture medium of cancer cells. These growth factors are assumed to be present in the culture supernatants of fibroblasts and may exert a paracrine effect on the proliferation of cancer cells. We also examined the intrinsic role of HGF/MET and FGFs/FGFR in ESCC proliferation. In addition, we examined the inhibitory effect of lapatinib on ESCC cell lines and studied whether the fibroblast supernatants affect the inhibitory effect of lapatinib on ESCC cell proliferation. Finally, we tested whether the FGFR inhibitor PD-173074 could eliminate the rescue effect against lapatinib that was induced by fibroblast supernatants.The addition of fibroblast supernatant induces cell proliferation in the majority of cell lines tested. The results of experiments to evaluate the effects of adding growth factors and kinase inhibitors suggests that the stimulating effect of fibroblasts was attributable in part to HGF/MET or FGF/FGFR. The results also indicate diversity in the degree of dependence on HGF/MET and FGF/FGFR among the cell lines. Though lapanitib at 1 μM inhibits cell proliferation by more than 50% in the majority of the ESCC cell lines, fibroblast supernatant can rescue the growth inhibition of ESCC cells. However, the rescue effect is abrogated by co-treatment with FGFR inhibitor.These results demonstrate that cell growth of ESCC depends on diverse receptor tyrosine kinase signaling, in both cell-autonomous and cell-non-autonomous manners. The combined inhibition of these signals may hold promise for the treatment of ESCC. Currently, esophageal cancer is the eighth most common cancer in the world ,2. EsophCancers are the end-product of accumulated effects of many genetic alterations, and the specific combination of changes is reflected in the unique characteristics of each tumor. The microenvironment of cancer cells has recently been shown to strongly influence the biologic properties of cancer .A tumor consists of a dynamic mixture of tumor cells, fibroblasts, endothelial cells, immune cells and extracellular matrix. In many solid tumors, the stroma has been recognized to be important in promoting tumor proliferation, invasion, metastasis, and chemo-resistance ,15. The Hepatocyte growth factor (HGF) regulates cell growth, cell motility, and morphogenesis by activating a tyrosine kinase signaling cascade after binding to the c-Met receptor . HGF is Fibroblast Growth Factors (FGFs) have been implicated in the regulation of cell differentiation, proliferation, migration and survival in many different cell types . The bioLapatinib is a dual tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR) and human EGFR-2 (HER2) tyrosine kinase domains ,29. ReceTo date, however, few studies have evaluated the role of stromal fibroblasts in ESCC. In this study, we focused on the relationship between ESCC cells and fibroblasts, the main component of cancer stroma. We prepared in vitro experimental systems to evaluate the interactions between ESCC and fibroblasts and clarify the mechanisms by which fibroblasts control proliferation of ESCC cells at the molecular level. We also studied the effects of various tyrosine kinase inhibitors to gain insight into new treatment strategies for ESCC.PHA-665752 and PD-173074 were obtained from Tocris Bioscience . Lapatinib was obtained from Bio Vision . Recombinant human HGF, recombinant human FGF1, 7, and 10 were obtained from R & D System . Stock solutions of PHA-665752, PD-173074 and lapatinib were prepared in dimethyl sulfoxide and stored at −80°C until use. Stock solutions of HGF and FGFs were prepared in phosphate-buffered saline (PBS) and stored at −80°C until use.2.We used 22 esophageal squamous cell lines: TE-1, TE-4, TE-5, TE-6, TE-8 TE-9, TE-10, TE-11, TE-14, TE-15, and EC-GI-10 were obtained from RIKEN Cell Bank , while KYSE30, KYSE50, KYSE70, KYSE140, KYSE150, KYSE170, KYSE180, KYSE220, KYSE270, T.T, and TTN were obtained from Health Science Research Resources Bank . All cancer cell lines were maintained in RPMI 1640 supplemented with 10% fetal bovine serum , glutamine, 100units/ml penicillin, and 100 μg/ml streptomycin, in a humidified atmosphere with 5% COPrimary human esophageal fibroblasts designated as HEF75 , HEF21113 pieces. Several pieces were placed in a six-well plate, and the explants cultured for 48 hours in DMEM  supplemented with 10% FBS, antibiotics, and glutamine at 37°C in a humidified atmosphere with 5% CO2. After removing the explants and non-adherent cells, the remaining cells were incubated for 1–2 weeks. The adherent cells were then trypsinized and passaged into a new culture flask at a ratio of 1:3 for further expansion. The cells were used for subsequent experimental study after the third passage.To isolate fibroblasts ,36, epitHuman lung-derived fibroblasts, HFL-III, obtained from RIKEN Cell Bank, were also used in some experiments.th and 25th passage after viral transduction were used. Immortalized human esophageal fibroblasts were designated as human esophageal fibroblast (HEF) 75-human telomerase reverse transcriptase (HEF75-hTERT).Immortalized fibroblasts, designated as HFE75-hTERT (human telomerase reverse transcriptase), were described previously . In brieRabbit polyclonal anti-phospho-Akt, anti-phospho-Erk antibodies  and goat polyclonal anti-beta actin  were used in this study.http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE63941).A comprehensive gene expression analysis was performed using an oligonucleotide microarray  as described previously . The dat3VO4 with a cocktail of proteinase inhibitors. After sonication, lysates were immersed in water at 98°C for five minutes and cleared by centrifugation. Protein concentrations were determined using the DC Protein Assay kit (BioRad). For Western blot analysis, equal amounts of protein samples were size-separated on 8% polyacrylamide gels and electroblotted onto a nitrocellulose membrane. Nonspecific binding was blocked by immersion of the membranes for 20 minutes in 5% skim milk in Tris-buffer saline at room temperature.Cells were lysed in a lysis buffer consisting of 20 mmol/L Tris–HCl (pH7.4), 150 mmol/L NaCl, 50 mmol/L NaF, and 1 mmol/L NaMembranes were washed with Tris-buffer saline buffer containing 0.1% Tween 20, incubated for one hour at room temperature with primary antibodies, washed, and then reacted with peroxidase-conjugated secondary antibodies. The antigen was detected using ECL Western Blotting Detection Reagents (Amersham) following the manufacturer’s instructions.3 cells) were plated in 96-well microtiter plates. After 24 hours, a supernatant of fibroblasts or growth factors , was added to the wells as appropriate.Cell viability was measured using the Cell Counting Kit (CCK) 8 assay  according to the manufacturer’s instructions. ESCC cells , PHA-665752 (MET inhibitor), PD-173074 (FGFR inhibitor), or 0.1% of dimethyl sulfoxide .At the end of the four-day incubation, the absorbance of each well at 450 nm was measured with a reference at 630 nm using a BIO-RAD model 680XR microplate reader .10 (concentration of reagents).The percentage of cell viability was calculated by following formula:%cell viability = (mean absorbance in test wells)/ (mean absorbance in control well) ×100. Results were plotted as cell viability versus logTo describe the blocking effect of PHA-665752 and PD-173074 on the growth promoting effect of fibroblast supernatant, a blunting index was calculated by the formula: blunting index (%) = 100-(fold change in the presence of PHA-665752 (or PD-173074) and fibroblast supernatant/fold change in the presence fibroblast supernatant alone) × 100.Human HEF75, HEF75-hTERT, and some cancer cells in a confluent state were cultured for 24 hours in RPMI/DMEM containing 0.1%FBS, and the supernatant concentration was measured by Quantikine® human FGF 7 immunoassay or human HGF immunoassay (R&D Systems). Measurements were performed in accordance with the manufacturer’s instructions.Analysis of variance was used to compare mean values of continuous variables among three or more groups, and P-values were calculated. When the P-value was less than 0.05 and the null hypothesis was rejected, Dunnett’s test, a method for post hoc comparison, was used to compare the groups. The Jmp9  statistical software package was used for analysis.The effect of fibroblast supernatant was evaluated to determine whether paracrine signaling induced by fibroblasts can influence the proliferation of cancer cells. Using the cell count assay, fibroblast supernatant from HEF75 and HEF75-hTERT increased cell proliferation by 1.25 fold or more in 12 and 21 of 22 cell lines, respectively  and purchased lung-derived fibroblasts (HFL-III), generally similar trends are observed (data not shown). These findings imply that growth factors or cytokines secreted by fibroblasts play an important role in cancer cell proliferation.We hypothesized that HGF and FGFs secreted by fibroblasts may be responsible for the stimulatory effect of fibroblast supernatant ,24,27,40Next, we examined the effects of adding growth factors HGF, FGF1, FGF7, and FGF10, to the culture medium of cancer cells. These growth factors, assumed to be present in the culture supernatants of fibroblasts, may exert a paracrine effect on the proliferation of cancer cells ,24,27,40These results indicate that cell proliferation of many ESCC cell lines is dependent on HGF/MET and FGF/FGFR either in an extrinsic or intrinsic manner. These experiments also indicate diversity in the degree of dependence on HGF/MET and FGF/FGFR among the cell lines tested, with more cell lines exogenously dependent on FGF/FGFR than on HGF/MET.Both Akt and ERK1/2 participate in the biological effects of growth factors -43. The Cell lines with proliferation induced by fibroblast supernatants  were selected. Western blot analysis was performed to assess the phosphorylation of AKT and ERK1/2 6 hours after adding supernatant. As shown in Figure To investigate the relative contribution of HGF/MET and FGFs/FGFR to the stimulatory effect of fibroblast supernatant, we tested the effect of PD-173074 (FGFR inhibitor) and PHA-665752 (MET inhibitor) treatment on cell proliferation induced by fibroblast supernatant. The results are shown in Figure Testing all ESCC cell lines for the relative contribution of HGF/MET and FGF/FGFR in a similar experiment, demonstrates that four cell lines  are dependent on HGF/MET, while eight cell lines  are dependent on FGF/FGFR. These results indicate that paracrine stimulation by fibroblast is dependent, at least in part, on HGF/MET or FGF/FGFR. These results also show that there is diversity among ESCC cell lines with regard to dependence on HGF/MET and FGF/FGFR.Cancer treatment strategies are currently shifting to targeted therapies. Several drugs are target members of the human epidermal growth factor receptor (EGFR/HER) family . This faIn this study we examined the inhibitory effect of lapatinib on ESCC cell lines. In addition, we studied whether the fibroblast supernatants affect the inhibitory effect of lapatinib on ESCC cell proliferation. The results are shown in Figure Recent studies indicate that paracrine stimuli from stromal fibroblasts may confer resistance to various molecular targeted agents including lapatinib -51. We rThe results of these experiments are shown in Figure The concentrations of HGF in culture supernatants of the fibroblasts HEF75, HEF75-hTERT, and HFL-III were 4049 pg/mL, 9390 pg/mL, and 9381 pg/mL, respectively, and the concentrations of FGF7 were 249 pg/mL, 1087 pg/mL, and 17 pg/mL, respectively. Culture supernatants of HFL-III, a lung-derived fibroblast line, contained high concentrations of HGF, but very low concentrations of FGF7. The culture supernatants of measured cancer cells  contained virtually no HGF or FGF7, suggesting that the autocrine activity of these cells was minimal.The importance of the tumor microenvironment for tumor growth has been recognized for many years . CancersTo date, few studies have comprehensively evaluated the interactions between ESCC and stromal fibroblasts. We studied 22 ESCC cell lines, the maximum available commercially, because many previous studies used few cell lines that were appropriate for the experiments. Furthermore, we isolated esophageal stromal fibroblasts from resected specimens and immortalized a portion of them. These studies clarify the roles of fibroblasts in the proliferation of ESCC and in resistance to lapatinib, given the diversity of cancer.We first examined cell proliferation induced by fibroblasts. The addition of fibroblast supernatant induced cell proliferation in the majority of ESCC cell lines. In particular, supernatants from immortalized fibroblasts had a stronger impact on cancer cell lines. HEF75-hTERT, immortalized fibroblasts, increased cell proliferation by 1.25 fold or more in 21 of 22 cell lines, while normal fibroblasts increased proliferation in 12 of 22 cell lines , fibroblast supernatant restored the growth of cancer cells inhibited by lapatinib, but the subsequent addition of PD-173074 abrogated the rescue effect of fibroblast supernatant against lapatinib Figure . These fWe studied 22 ESCC cell lines taking into account the diversity of malignancies. In more than half of the cancer cell lines tested, proliferation was induced by culture supernatants of immortalized fibroblasts. Growth factors, such as HGF or FGFs secreted by fibroblasts influence cell proliferation and resistance to lapatinib in some cancer cell lines, although the degree of dependence differs among the cell lines. The addition of fibroblast supernatant attenuates the effect of lapatinib. However, the rescue effect of fibroblast supernatant is abrogated by combining an FGFR inhibitor with lapatinib. Although this study was performed in vitro, these results may lead to the development of new treatment strategies.


Mosquitoes are highly effective vectors for transmission of human and animal pathogens. Understanding the relationship between pathogen and vector is vital in developing strategies to predict and prevent transmission. Cell lines derived from appropriate mosquito hosts provide a relatively simple tool for investigating the interaction between the host and viruses transmitted by mosquitoes. This review provides a brief overview of the development of mosquito cell lines, methods of isolation, their availability and application for investigating insect-virus interactions. Anopheles mosquitoes and results in the greatest mortality predominantly in Sub-Saharan Africa. In 2010, the World Health Organization estimated that there were 219 million cases  of malaria with 3.3 billion people at risk. Dengue is transmitted by Aedes mosquitoes and the incidence has grown dramatically with 50–100 million cases/year and 2.5 billion people at risk. Significant outbreaks of other mosquito-borne diseases such as Chikungunya, Japanese encephalitis and lymphatic filariasis impose a substantial burden on global health and economics in developing countries. Arboviral diseases transmitted by mosquitoes are a driver for poverty in much of the developing world and have shown an increase in incidence in the past few decades with major outbreaks occurring in previously non-endemic areas [Mosquitoes are responsible for the transmission of numerous infectious diseases including malaria, dengue fever, West Nile fever and Japanese encephalitis. Malaria is transmitted by The mosquito lifecycle in its simplest form is composed of a series of life stages beginning with eggs laid on or near water that hatch after a number of days into larvae. The larvae obtain nutrition predominantly through filter-feeding but predation on other larvae and small invertebrates also occurs for certain species. Mosquitoes develop through four instar phases, to form non-feeding pupae, which metamorphose into adults Figure . Adult mIn vitro studies using mosquito cell lines can be carried out to provide valuable information on aspects of these interactions. Here we review the history, isolation, availability and application of mosquito cell lines, and how cell lines can be used to contribute to understanding of the potential vector competence of UK mosquitoes for arboviruses.In recent years, interest has developed in a number of areas associated with virus-mosquito interactions. One key area of research has been elucidation of the immune response of insects against pathogens, and viruses in particular. In contrast to vertebrates, which have developed the interferon system to suppress virus replication, insects have an alternative innate immune mechanism commonly referred to as RNA interference (RNAi). This immune mechanism in mosquitoes has been reviewed extensively in recent years , 5. BrieAnopheles (An.), Culex (Cx.) and Aedes (Stegomyia) (Ae.) genera and several cell lines have been established. Table Ae. aegypti[Ae. aegypti CCL-125 cell line was initially characterized as not susceptible to DENV [Ae. aegypti in the late 1980s [Ae. aegypti cell lines such as RML-12 have been shown to be particularly susceptible to infection with DENV [There are over 500 insect cell lines now established from numerous insect orders including Diptera, Lepidoptera and Hemiptera, derived from different tissue sources , 8. The  aegypti. The Ae. to DENV . However to DENV . The Aagte 1980s . Subsequith DENV  and numeith DENV .Table 1Ae. albopictus, is a competent vector of many arboviruses including DENV, Chikungunya virus [Ae. albopictus, such as the C6/36 cell line , were generated from larvae in the mid 1960s [Ae. albopictus and has been shown to synthesize hormone-inducible proteins and thus forms a useful model for understanding hormone responses in insects [The Asian tiger mosquito, ya virus  and Eastya virus . The firid 1960s . Lineageid 1960s . C6/36 c insects .Ae. pseudoscutellaris is a vector of subperiodic Wuchereria bancrofti, which is responsible for cases of human lymphatic filariasis in the South Pacific. A number of cell lines have been derived from larvae of this species in the 1970s, and were shown to be susceptible to a range of arboviruses [Ae. pseudoscutellaris cell line, AP-61, was established and shown to be susceptible to DENV infection [Aedes species, Ae. triseriatus, is a vector of La Crosse virus (LACV), which is a cause of encephalitis in the Midwest of the USA, and has been shown to be an efficient laboratory vector of EEEV. Three cell lines  were established from Ae. triseriatus embryos or larvae in the mid 1990s [oviruses . In partnfection , 22. Anoid 1990s , and theAe. vexans mosquitoes are widespread throughout Europe and are potential vectors of WNV and RVFV [Ae. vexans[and RVFV , 25. A c. vexans but suscAnopheles mosquitoes, particularly species in the An. gambiae complex, are responsible for malaria in Sub-Saharan Africa. Malaria has the highest mortality rate of any mosquito-borne disease and is justifiably the main focus of research on mosquitoes as vectors of disease. Anopheles cell lines have been generated including An. gambiae Mos.55 [in vitro model for the study of both the humoral and cellular immune defence of An. gambiae mosquitoes. An. stephensi mosquitoes are the primary vectors of malaria in South Asia, and several cell lines have been generated from larvae including Mos.43 [An. albimanus is a major malaria vector in Central and South America, and the LSB-AA695BB cell line has been established from An. albimanus embryos [e Mos.55 , Sua1B ae Mos.55 . The 4a-g Mos.43  and MSQ4g Mos.43 . An. alb embryos .Culex mosquitoes that are responsible for transmission of human diseases. Cx. quinquefasciatus is a member of the Cx. pipiens complex and is the primary vector of Bancroftian lymphatic filariasis in tropical and sub-tropical regions. It is also the main vector of WNV in the USA and Europe. A cell line was established from this species using ovaries as the source tissue in the 1960s [Cx. quinquefasciatus cell line has been established from embryos [Cx. theileri is a natural vector of Dirofilaria immitis (dog heartworm) in southern Europe and is thought to be the major vector of Rift Valley fever virus (RVFV) in parts of southern Africa. A cell line was established for this vector species using embryos as the source material and was shown to be susceptible to a number of arboviruses [Cx. tritaeniorhynchus mosquitoes are the principal vectors of JEV in Asia, and cell lines have also been established from embryos of this species [Cx. tritaeniorhynchus NIID-CTR cell line is highly susceptible to both JEV and DENV infection, and provides a valuable model for virus replication in the host [Cx. bitaeniorhynchus, a mosquito found throughout Asia and a potential vector of JEV, has been used to generate cell lines that are susceptible to several arboviruses [There are a number of he 1960s , and mor embryos . Cx. theoviruses . Cx. tri species , 36. Theoviruses .Toxorhynchites amboinensis has no known vector competence for human pathogens as this mosquito is unable to blood feed. This species has been used in mosquito biocontrol strategies, since the large predatory larvae eat other mosquito larvae such as Ae. aegypti. However, a cell line derived from Toxorhynchites amboinensis, TRA-171, has been established and shown to be susceptible to DENV infection, although the significance of this observation remains unclear [ unclear .in vivo vector competence. For example, cell lines could be used to provide some preliminary information on the susceptibility of midgut or salivary gland cells prior to in vivo transmission studies. Embryos are now commonly used as the source for mosquito cell cultures as they contain cells with the potential to differentiate into larval and adult tissues, resulting in a wide diversity of cell morphologies.The source material for primary insect cell line culture is an important consideration. Ovaries were the first insect tissues used throughout the 1960s and 1970s, predominantly with Lepidoptera. Adult female mosquito tissues such as salivary glands or midguts could be used to generate cell lines that would be relevant to specific stages of virus-mosquito interactions that influence ■ The protocol for cell culture generation from either embryos or female tissues such as ovaries requires adult female mosquitoes (4-5 days old) to be blood fed using standard procedures. Mosquito eggs are collected 4 days post-blood feed, when an egg laying pot is placed into the cage containing gravid females.■ Mosquito eggs are transferred from egg laying pots and rinsed thoroughly in 70% ethanol and then sterile water to prevent any bacterial contamination from the egg chorion (mosquito eggs are sufficiently impervious to simple disinfectants such as 70% ethanol). If tissues of adult female mosquitoes are used as the source material, tissues are dissected using sterile procedures.■ Embryos or tissues are mechanically disrupted in insect cell culture media using a tissue homogenizer such as a plastic pestle. Additional methods have been used to disrupt embryonic cells such as the use of bleach or using enzymes including trypsin. However, care must be taken to avoid using chemicals that could potentially inhibit cell growth.■ Insect cell culture media can vary for different mosquito species but standard formulations include Grace’s, Schneider’s and Mitsuhashi and Maramorosch media. A typical cell medium for mosquito cells is supplemented with fetal bovine serum and the antibiotics penicillin/streptomycin to prevent bacterial contamination.■ The cell medium containing crushed mosquito material is then transferred to tissue culture flasks. Additional cell culture medium is added and cells are incubated at 26°C until cell attachment occurs. Culture medium is replaced every 3-4 days by vigorously shaking the tissue culture flasks and replacement with fresh medium.■ Subcultures are obtained by passaging cells continually through disrupting confluent cell monolayers and transferring cells in suspension to a new tissue culture flask with fresh medium. Observation of cell morphology, division and growth with an inverted microscope is used to generate several subcultures.A brief protocol for derivation of cells from mosquito embryos or tissues is detailed below.A large number of embryos can be obtained from laboratory mosquito colonies to provide source material for multiple cell line culture experiments. An important consideration in the use of embryos for cell culture is the timing of embryogenesis. An incubation period after egg laying of 16-20 hours  was shown to be optimal for the successful generation of mosquito cell lines , 33. A cAe. aegypti, Ae. albopictus and Toxorhynchites amboinensis (http://www.lgcstandards-atcc.org). The An. stephensi MSQ43 cell line is available from the Malaria Research and Reference Reagent Resource Center (www.mr4.org). Requests for mosquito cell lines can also be sent to the World Reference Centre for Emerging Viruses and Arboviruses (http://www.niaid.nih.gov/labsandresources/resources/dmid/wrceva).The majority of cell lines currently used in arboviral research are obtained through direct contact with laboratories currently working with that particular cell culture. Only a few mosquito cell lines are commercially available. For example, the American Type Cell Collection only lists three cell lines derived from In vitro serial cell line passage experiments can be used to determine the host range of arboviruses in mosquitoes, ticks and vertebrates [in vivo infections [Ae. aegypti to DENV infection has also been characterized in the immune-competent Aag2 cell line. Using transcriptomic analysis, it has been shown that DENV is capable of actively suppressing the mosquito immune response in infected cells [tebrates , 40. In tebrates . Furtherfections . The anted cells . Similared cells .In vitro studies could be used to provide preliminary evidence for the potential of some mosquito species and vertebrate hosts to support replication of emerging arboviruses. For example, Rabensburg virus (RABV), a Flavivirus isolated from Cx. pipiens, was first considered to be a potential new lineage of WNV [Cx. pipiens mosquitoes support replication of RABV, peroral transmission of infectious RABV was much lower, and vertical transmission higher, compared to WNV. Additionally, experimentally inoculated avian hosts did not become infected [in vitro vector-virus interaction may not always reflect in vivo vector competence or disease transmission in mosquito field populations.e of WNV , but it e of WNV . RABV die of WNV . Furtherinfected . Howeverin vivo transmission assays that involve collection of mosquito saliva during expectoration. Initially cell lines are used to amplify arbovirus titers required for oral feeding experiments. Mosquitoes presented with an arbovirus-infected blood meal undergo ‘forced salivation’, typically 7-14 days post-infection, to measure the length of the EIP of arboviruses within mosquitoes. Following forced salivation, mosquito cell lines such as C6/36 are inoculated with the collected saliva. Incubation of cells is then followed by an antibody-based detection protocol to determine whether infectious virus is present in saliva or through the appearance of plaque forming units. For low virus titers in saliva, several rounds of cell line passage may be undertaken to increase sensitivity.Mosquito cell lines are also used extensively to passage arboviruses for Drosophila) or human cell lines. Mosquitocidal cytotoxins were shown to be toxic to mosquito cells but not to Drosophila cells [Bacillus thuringiensis israelensis (Bti) produces insecticidal toxins active against mosquitoes that can be an effective larvicide for mosquito biocontrol. Cry4B is one of the major toxins produced by Bti and Aedes cell lines were used to demonstrate that Cry4B binds to several midgut membrane proteins including prohibitin, a protein recently identified as a receptor for entry of DENV into Aedes cells [Aedes cells to show that pre-exposure to Cry4B results in a significant reduction in the number of infected cells compared to mock-exposed cells.Mosquito cell lines have also been used to determine the efficacy of novel compounds such as insecticides and biocontrol agents. Cell-based screening platforms have been developed to identify new compounds that are lethal to mosquito cell lines but show little or no activity against other insects  strain SPFLD-MO-2011-MP6 was isolated in C6/36 cells from a pool of male Ae. albopictus mosquitoes that were reared to adults from larvae collected in the USA [Ochlerotatus caspius mosquitoes was identified in Portugal and is referred to as Ochlerotatus flavivirus (OCFV) [Mosquito-only flaviviruses (MOFs) have no known vertebrate reservoir host and have only been identified in mosquitoes. Cell lines have played an important role in the isolation and characterization of MOFs. The cell fusing agent virus was isolated in 1975 from an ell line . Additiorhynchus, Kamiti cintoshi, 52, Naafricana and Calbtarsalis–56. Six squitoes . Inoculato cells . Recentl the USA . AEFV dos (OCFV) . OCFV wain vitro studies. Culicoides midges are important vectors of arboviruses such as bluetongue virus (BTV). Studies in Culicoides KC cell lines have demonstrated a functional RNAi response that inhibits BTV infection [Along with mosquitoes, other arthropods may also provide a useful source of cell lines for nfection . Tick cenfection .An. gambiae  and Ae. aegypti (dengue), further cell lines are needed for native temperate mosquito species that are considered potential vectors of arboviruses. Sporadic WNV outbreaks have occurred in warmer regions of Europe for the past 20 years and could occur more frequently as a result of climate change [Cx. pipiens mosquitoes are widespread throughout Europe, and are critical vectors of WNV in the USA and southern Europe. Currently available cell lines, such as C6/36 and RML-12, are high passage cell lines, which may not be susceptible to many arboviruses that are considered a threat to countries such as the UK, including WNV. Ae. vexans is a vector of Tahyna virus and is the most common mosquito in Europe, but is relatively rare in the UK. This species has also been shown to be a competent vector of both WNV and RVFV under laboratory conditions. Preliminary experiments have been undertaken to generate an Ae. vexans cell line from a colony of UK origin. As shown in Figure Ae. vexans to a diverse range of arboviruses that are considered to be a potential threat to the UK and help to inform future in vivo vector competence studies.Although several mosquito cell lines have been established from major mosquito disease vectors such as e change . Cx. pipCx. pipiens, which are potential WNV bridge vectors from birds to humans [Ae. vexans and Cx. modestus, have been shown to transmit WNV in other countries. Any change to the UK climate that causes an increase in mean temperature or prolonged periods of above-average warm weather may potentially increase the geographical distribution of European mosquito-borne diseases such as WNV. As a result, studies to determine the vector competence of mosquito species that are most likely to serve as bridge vectors for WNV are required to assess the likelihood of introduction and transmission in the UK. Investigation using mosquito cell lines will therefore provide a relatively simple method for deriving preliminary data on a range of virus-vector interactions with UK mosquito species. Cell lines that are shown to not be susceptible to certain viruses likely indicate that in vivo vector competence will be limited for that mosquito species. The genetic background of mosquito host vectors influences the vectorial capacity. Specific vector genotype x virus genotype (G x G) interactions may promote adaptation of viral lineages to local mosquito vector genotypes [There are over 30 endemic mosquito species in the UK and several, including o humans . Furtherenotypes . The virenotypes . Virulenenotypes . Passingenotypes . The evoenotypes . Howeverenotypes . MosquitCx. pipiens established from Colorado, USA, has been shown to be infected with a Culex flavivirus (CxFV) and early suppression of WNV replication was observed [Cx. quinquefasciatus mosquitoes [Cx. pipiens and Ae. vexans mosquitoes would provide suitable models to investigate insect-specific flaviviruses that are present in UK populations. Infection of cell lines with arboviruses such as WNV would provide valuable information to inform vector competence studies of these native mosquito species.Experiments to determine virus-host interactions, with an appropriate cell line of UK genetic background, are necessary to assess the likely impact of the introduction of arboviral transmission in a new area and the ability of the pathogens to become established in the local mosquito population. The presence of insect-specific flaviviruses, particularly in mosquito populations, may also influence transmission of arboviruses to humans. For example, a laboratory colony of observed . Howeversquitoes . Establi


PETF in Chlamydomonas decreased the reactive oxygen species (ROS) level and enhanced tolerance to heat stress. In addition to PETF, an endogenous anaerobic induced FDX5 was overexpressed in transgenic Chlamydomonas lines here to address the possible functions of FDX5. All the independent FDX transgenic lines showed decreased cellular ROS levels and enhanced tolerance to heat and salt stresses. The transgenic Chlamydomonas lines accumulated more starch than the wild-type line and this effect increased almost three-fold in conditions of nitrogen depletion. Furthermore, the lipid content was higher in the transgenic lines than in the wild-type line, both with and without nitrogen depletion. Two FDX-overexpressing Chlamydomonas lines were assessed in a photo microbial fuel cell (PMFC); power density production by the transgenic lines was higher than that of the wild-type cells. These findings suggest that overexpression of either PETF or FDX5 can confer tolerance against heat and salt stresses, increase starch and oil production, and raise electric power density in a PMFC.Ferredoxins (FDX) are final electron carrier proteins in the plant photosynthetic pathway, and function as major electron donors in diverse redox-driven metabolic pathways. We previously showed that overexpression of a major constitutively expressed ferredoxin gene  Photosynthetic microalgae are autotrophic cells that have rapid growth capacity; as such they are key resources in biorefineries . HoweverChlamydomonas reinhardtii has been used as a model organism for studying photosynthesis, starch metabolism and oil synthesis -hydrogenase HydA1, so the role of FDX5 in the anaerobic pathway is unclear [In d FDX2-6 . These Fd FDX2-6 ,26. PETFd FDX2-6 . PETF and FDX2-6 ,25. The d FDX2-6 ,25. FDX5 unclear ,27.Chlamydomonas cell lines overexpressing either PETF or FDX5 were generated to clarify whether increasing gene expression levels of FDXs could alleviate oxidative damage under heat and salt stresses. Starch content was evaluated in these transgenic lines. Lipids accumulated more in the transgenic lines than in the wild-type cells, irrespective of nitrogen depletion. The two FDX overexpressing Chlamydomonas lines were applied in PMFCs; power density production by the transgenic lines was higher than that for the wild type.In this study, transgenic PETF and FDX5, fused downstream of the B2T (β2-tubulin) promoter, were transformed into Chlamydomonas by electroporation, and at least three putative independent transgenic Chlamydomonas lines were selected for each construct. To evaluate the putative transgenic Chlamydomonas lines, PCR-based genomic DNA analysis was performed using specific primers to confirm the presence of transgenes. The transgene PETFtg was detected in the cell lines P22, P51 and P67, and FDX5tg was detected in lines F5-1, F5-81, F5-302 and F5-303, respectively. In contrast, no transgenes were detected in the wild-type line  stress have been reported to increase the levels of ROS and inhibit the cell growth in ydomonas . To exam 80%–90% B. Taken Chlamydomonas [FDXs increased starch accumulation under nitrogen starvation, we measured the starch contents of PETF and FDX5 overexpressing transgenic lines after nitrogen starvation for various periods. The starch content increased by 60% relative to wild type in transgenic lines overexpressing either PETF or FDX5 in normal growth conditions   A. Meanwh mW·m−2) A. Furtheay three B. After ee weeks B, and liell line B.2O2 [Sybechocystis sp., has been proposed to play a role in detoxification of oxidative compounds [PETF, a major form of ferredoxin, decreases the ROS level and contributes to tolerance to heat stress in Chlamydomonas [Chlamydomonas transgenic lines overexpressing either PETF or FDX5 were significantly reduced, resulting in improved tolerance to heat and salt stresses. Our investigation supports the hypothesis described above that the overexpression of FDXs decreases ROS levels and contributes to tolerance to abiotic stresses in Chlamydomonas.Microalgae are considered a primary resource for third-generation biofuels, because they can produce biomass rapidly with simple cultivation requirements. Some microalgal strains can even be directly cultivated with flue gas and wastewater ,32. In o2O2 ,21,36,372O2 . Flv3, aompounds . Previouydomonas . Proteinydomonas , implyinChlamydomonas by RNAi technology results in a higher resistance to photodamage, and increases light penetration properties compared to that in non-transgenic cells [PETF overexpression lines may transport excess electrons from photosystems more effectively than the nontransgenic line, reducing ROS levels and also preventing photoinhibition, thereby increasing the starch content. Given that FDX5 interacts with PETF, the results described in the discussion above in FDX5 overexpression lines may be mediated by PETF.Microalgae have chloroplasts that contain light-harvesting antenna complexes (LHCs) to capture light energy, and chloroplasts generate ROS during energy transfer steps of photosynthesis . Down-reic cells . FDXs acic cells . In the Chlamydomonas overexpressing either the PETF or the FDX5 gene all showed higher starch accumulation than the wild type. In Chlamydomonas, PETF, the most abundant ferredoxin, has been shown to interact directly with FNR1, which can reduce NADP+ to the NADPH that is later used in the Calvin Cycle to assimilate CO2 [In this study, transgenic lines of late CO2 ,42. Morelate CO2  involvedlate CO2 ,44. Bothlate CO2 .2 assimilation, in turn, to increase lipid accumulation. Moreover, in vitro activity analyses indicated that FDXs provide electrons to fatty acid desaturases [PETF- and FDX5-overexpressing lines in future work.Several enzymes, such as pyruvate dehydrogenase and fatty acid synthase, require ATP and NAD(P)H to run fatty acid biosynthesis . The traaturases ,46. We wPETF and FDX5 overexpression transgenic lines were applied in a PMFC and the electric power density was higher than for wild-type cells, indicating that these FDX transgenic lines have high electrochemical activities. It is possible that overexpression of FDXs can enhance electron transfer to the anode of the PMFC. Another possibility is that the FDX transgenic lines accumulate more biomass, such as starch, which can be converted into cytosolic NADPH by the pentose phosphate pathway. In turn, electrons transfer to the extracellular space by NADPH oxidases encoded by RBO1 (respiratory burst oxidase) and RBO2 [et al., 2015). Interestingly, the FDX5 transgenic line exhibited higher electric power density than the wild-type line and PETF transgenic lines, implying that FDX5 is a major electron carrier in Chlamydomonas in anaerobic conditions when FDX5 is induced and FDX5 is stabled [Microbial fuel cells (MFCs) are a microbial electricity generation system that produces electric power from catalytic reactions of organic substrates and biomass. The design of better materials for MFCs, the optimization of conditions for growth and activity of microbial cells, and the screening of more electrochemically active microbes, are all areas of work that have been undertaken in a bid to enhance the generation of electric power by MFCs ,50,51,52and RBO2  (Anderso stabled . Future Chlamydomonas reinhardtii (CC125) and transgenic PETF or FDX5 overexpression lines were grown in Tris-acetate-phosphate (TAP) liquid medium [−2·s−1) at 25 °C. The cell density was adjusted to 3 × 106 mL−1, and 1 mL of cell suspension was added into 99 mL of TAP with shaking at 150 rpm for liquid culture.The wild-type strain of d medium  for a 12FDX5 gene, the FDX5 cDNA fragment was amplified by reverse transcription polymerase chain reaction (RT-PCR) with primers cFDX5-F: 5ʹ-ACGGGCCCATGCTGTGCGCGCGCTCCCAG-3ʹ (ApaI site underlined) and cFDX5-R: 5ʹ-GCCATATGTTACTGGTGCTTGCCGTACTCG-3ʹ (NdeI site underlined). The PCR fragment was digested with ApaI and NdeI, and ligated into pHYG3-PETF [2-tubulin promoter driving expression of the FDX5 gene. As a terminator, the 3ʹ-UTR of the rbcS2 gene  was amplified by PCR with rbcS3-F: 5ʹ-AACATATGCGCTCCGTGTAAATGGAGGC-3ʹ (NdeI site underlined) and rbcS3-R: 5ʹ-AACATATGTCTAGACGCTTCAAATACGCCCAGC-3ʹ (NdeI site underlined), digested with NdeI and ligated with pHYG3-FDX5 to produce pHYG3-FDX5-R. The same construction strategy was applied to produce plasmid pHYG3-PETF-R for expression of PETF.To construct a plasmid for constitutive expression of the YG3-PETF  to generC. reinhardtii strain CC125 cells were electroporated according to a method described previously [ScaI and then transferred into a 4 mm gap electroporation cuvette, respectively. For electroporation, the Gene Pulser Xcell™ system  was used, with parameters 2 kV·cm−1, 25 µF, and 500 Ω. Cells were screened on TAP plates containing 20 μg·mL−1 hygromycin. All putative PETF and FDX5 transgenic cell lines were confirmed by genomic PCR with specific primers to amplify the recombinant DNA fragment including the β-tubulin (B2T) promoter, FDX coding sequence and the rbcS2 terminator region (RBCS 3ʹUTR). These primers were B2TP-F (5ʹ-CTAGATCACTACCACTTCTACACAG-3ʹ) and P721 (5ʹ-GCGTATCACGAGGCCCTTTC-3ʹ).eviously . Both pH6 mL−1 cells were cultured in TAP medium. One mL of cell suspensions were subjected to treatment at 42 °C under light (125 µmol photons·m−2·s−1) for various periods, and then recovered for 72 h under continuous light at 25 °C. Cell survival rate was determined by trypan blue staining analysis. The cell suspension was mixed with an equal volume of 0.4% (w/v) trypan blue solution. The survival rate was determined by hemocytometer and calculated as (unstained living cell number)/ × 100%.For heat stress treatment, log phase of cells were centrifuged and then 3 × 106 mL−1 cells were cultured in TAP medium containing different NaCl concentrations  under light condition (125 µmol photons·m−2·s−1) for 12 h. Cell survival rate was determined as described as heat stress treatment.For salt stress treatment, log phase of cells were centrifuged and then 3 × 10Chlamydomonas cells  using a plant total RNA kit . Then, cDNA fragments were synthesized from 1 μg of total RNA using the Transcriptor First Strand cDNA Synthesis Kit . To measure the total PETF and FDX5 transcripts in PETF-transgenic, FDX5-transgenic and wild-type lines, quantitative PCR was performed using a PikoReal™ 96 Real-Time PCR System . Primers PETF-F (5ʹ-GCTATGCGCTCCACCTTC-3ʹ) and PETF-R (5ʹ-CGTCCAGGATGTAGGTGTCA-3ʹ) were designed to amplify PETF transcripts. Primers FDX5-F2 (5ʹ-CGGGCAAGACGAAGACTATGG-3ʹ) and FDX5-R2 (5ʹ-GGGTAGGCGAGCACATGAG-3ʹ) were designed to amplify FDX5 transcripts. CBLP (G-protein beta subunit-like polypeptide) transcripts were analyzed as an internal control using primers Re-Cblp-F and Re-Cblp-R as previously described [Total RNA was extracted from log phase of escribed .2O2 measurement, an Amplex red hydrogen peroxide/peroxidase assay kit  was used. Log phase Chlamydomonas cells  were collected by centrifugation and then frozen in liquid nitrogen, ground with a steel ball, and dissolved in 100 μL of 1 × reaction buffer from the assay kit. The mixture was centrifuged at 13,000× g for 5 min, and then the supernatant was used to measure cellular H2O2 concentration after horseradish peroxidase treatment at 25 °C for 30 min. The H2O2 concentrations were determined using a standard curve plotted from 0.2 to 1.0 μM H2O2 standards. Data were collected with five repeats.For H2DCFDA) and then observed using a Zeiss LSM 510 META confocal laser-scanning microscope . Fluorescent signals emitted from H2DCFDA and autofluorescence of chlorophyll were excited at 488 nm, and emitted signals were obtained at 500–530 and 650–710 nm, respectively.To detect ROS, cells were incubated with 10 μM 2ʹ,7ʹ-dichlorodihydrofluorescein diacetate (H7Chlamydomonas cells in log phase were cultured in 100 mL TAP and nitrogen starved-TAP medium for various periods. Cells were harvested by centrifugation and then freeze-dried. Sample dry weight was measured and then cells were resuspended in 80% (v/v) ethanol at least three times to remove chlorophyll, judged by the loss of green color. Samples were dried again at 70 °C and resuspended in 50 mM HClO4 to measure starch contents, as described previously [Total 3 × 10eviously .7Chlamydomonas cells in log phase were cultured in 10 mL TAP and nitrogen starved-TAP medium for various periods. Cells were pelleted by centrifugation and frozen at −20 °C. Whole cells were thawed in 200 μL of saponification reagent (25% methanol in 1 N NaOH), lysed with a stainless-steel ball and then heated evenly in a boiling water bath for 30 min. For colorimetric detection of cellular lipids, 200 μL of neutralization reagent  and 200 μL of copper reagent ((1 M triethanolamine):(1 N-acetic acid):(6.45% (w/v) Cu (NO3)2·3H2O) = 9:1:10) were added to each sample, and then mixed with 250 μL of chloroform. After centrifugation, the organic phase was obtained and then subjected to relative lipid content measurement as described [To rapidly evaluate lipid levels, a colorimetric quantitation from algal cultures was applied as described , with slescribed .2) that were installed horizontally at opposite side of PMFC and a platinum wire was applied to connect the chambers to conduct electricity. Before the PMFC was set, the membrane and electrodes were pre-treated as described by Raman and Lan (2012); the electrodes were pre-treated with 1.0 M HCl for 1 h followed by 1.0 M NaOH for 1 h; the Nafion 211 membrane was pre-treated by sequentially boiling it in H2O2 (30% v/v) and deionized water, followed by soaking in 0.5M H2SO4 and then deionized water, each for 1 h. The anode chamber contained 3 × 108 harvested algal cells in TAP medium with 80 mM NaCl, and was continuously stirred at 250 rpm using a magnetic stirrer under anaerobic condition. The cathode solution contained 0.1 M K3Fe(CN)6, as an electron acceptor, with 0.5 M K2HPO4 and 80 mM NaCl at pH 7. The PMFC was operated under 4 h of light (~30 µmol photons·m−2·s−1) and 4 h of darkness. The voltage and current across the external resistor (1 kΩ) were measured at 1 min intervals using a dual-channel data collection multi-meter . Power density  was calculated as PD = IV/area, where P = power, I = current (A), V = voltage (V).A dual-chambered microbial fuel cell was fabricated using transparent poly-acrylic plastic plates. The two chambers were separated by a Nafion 211 membrane . The volume of each chamber was 70 mL. Two electrodes were graphite plates (each of 45.09 cm


Alterations in cell proliferation and tissue remodeling are major hallmarks of cancer. Given that these two physiological processes require a significant investment of metabolic resources, we hypothesize that the metabolism of cancer cells is, in a first approximation, determined by the magnitude of cell proliferation and tissue remodeling.in vitro data characterizing cancer cell lines and gene expression profiles of human cancers.To address this question in further detail we have analyzed in vitro data, cancer cell lines align from an extreme of highly proliferating cells of relatively small size to another extreme of slowly proliferating cells with large size and mesenchymal properties [in vitro data suggest that these different subtypes have a differential sensitivity to diverse drug classes targeting metabolism [We show that the metabolism of cancer cells is strongly correlated with cell proliferation and tissue remodeling. Based on operties . Similaroperties . The strtabolism .We conclude that human cancers can be subject to a universal cell-proliferation/tissue-remodeling classification independent of their site of origin that can guide personalized treatment targeting cancer metabolism.


CRC is a significant cause of cancer mortality, and new therapies are needed for patients with advanced disease. TAK-733 is a highly potent and selective investigational novel MEK allosteric site inhibitor.in vivo. Tumor growth inhibition index (TGII) was assessed to evaluate the sensitivity of the CRC explants to TAK-733 while linear regression was utilized to investigate the predictive effects of genotype on the TGII of explants.In a preclinical study of TAK-733, a panel of CRC cell lines were exposed to varying concentrations of the agent for 72 hours followed by a sulforhodamine B assay. Twenty patient-derived colorectal cancer xenografts were then treated with TAK-733 Fifty-four CRC cell lines were exposed to TAK-733, while 42 cell lines were deemed sensitive across a broad range of mutations. Eighty-two percent of the cell lines within the sensitive subset were BRAF or KRAS/NRAS mutant, whereas 80% of the cell lines within the sensitive subset were PIK3CA WT. Twenty patient-derived human tumor CRC explants were then treated with TAK-733. In total, 15 primary human tumor explants were found to be sensitive to TAK-733 (TGII ≤ 20%), including 9 primary human tumor explants that exhibited tumor regression (TGII > 100%). Explants with a BRAF/KRAS/NRAS mutant and PIK3CA wild-type genotype demonstrated increased sensitivity to TAK-733 with a median TGII of −6%. MEK-response gene signatures also correlated with responsiveness to TAK-733 in KRAS-mutant CRC.The MEK inhibitor TAK-733 demonstrated robust antitumor activity against CRC cell lines and patient-derived tumor explants. While the preclinical activity observed in this study was considerable, single-agent efficacy in the clinic has been limited in CRC, supporting the use of these models in an iterative manner to elucidate resistance mechanisms that can guide rational combination strategies. The RAS-RAF-MEK-ERK (MAPK) pathway is a major contributor to cell growth and survival and is frequently dysregulated in numerous cancers , 9. SignDue to the frequent aberration of this signaling cascade in malignant tissues, MEK has emerged as an attractive target in cancer. Inhibition of MEK impairs proliferation and affects a diverse array of cellular events including differentiation, apoptosis, and angiogenesis , 6. MEK 50 of 3.2 nM that selectively binds to and inhibits the activity of MEK1/2, preventing the activation of MEK1/2-dependent effector proteins and transcription factors. TAK-733 has demonstrated potent anticancer activity in several solid tumor mouse xenograft models and exhibited potent enzymatic and cell activity with an EC50 of 1.9 nM against ERK phosphorylation, the downstream target within the RAS/RAF/MEK/ERK pathway, in cells  and were then incubated overnight at 4°C in blocking buffer plus 0.1% Tween-20 with the primary antibodies . Blots were then washed 3 × 10 minutes in 1× TBS containing 0.1% Tween-20 and incubated with the appropriate secondary goat anti-rabbit and goat anti-mouse IgG (H + L) DyLight™ conjugated antibodies at 1:15,000  for 1 hour at room temperature. Following 3 × 10 minutes of washes, the blots were developed using the Odyssey Infrared Imaging System (LI-COR Biosciences). Immunoblot experiments were done in triplicate for each antibody and representative blots are depicted.3 (F1) at which point they were harvested, divided, and transplanted to an additional five mice (F2) to maintain the tumor bank. After a subsequent growth passage, tumors were excised, transplanted onto both flanks of nude mice, and expanded into cohorts of ≥25 mice for treatment. Tumors from this cohort were allowed to grow until reaching approximately 150 to 300 mm3, at which time they were equally distributed by size into the two treatment groups: control and TAK-733 treated. Because of the variability in take rates of the human patient explant material, enough mice were designated into each group based on the number of overall tumors (n = at least 12 tumors per group). Mice were treated for at least 28 days with either vehicle control (0.5% methylcellulose) or TAK-733 (1 mg/kg) once daily by oral gavage. Tumor growth inhibition index was calculated from average volume of the treated (Vt) and vehicle control (Vvc) groups, with the equation: TGII = 100 × /.PDX establishment and treatment protocols were conducted under previously described procedures , 34. BriRaw microarray gene expression data for the KRAS mutant CRC cell lines was obtained from the Cancer Cell Lines Encyclopedia (GSE36133). These samples were profiled by the Affymetrix HG-U133 Plus 2 arrays. Gene expression profiles were normalized by RMA and extracted using Affymetrix Power Tools (APT). Significant Analysis of Microarrays (SAM) (PMID: 11309499) was performed using R, with 500 permutations. Probe sets that passed FDR < 0.25 were selected as significantly differentially expressed in MEK sensitive and resistant KRAS mutant CRC cell lines. Raw microarray gene expression data for the CRC PDX models were profiled by the Affymetrix HuGene 1.0 arrays. Gene expression profiles were normalized by RMA and extracted using APT. Probe sets were matched between platforms using the BEST_MATCH probe sets provided by Affymetrix. Probe sets that matched between two platforms were Z-normalized independently and merged into single gene expression profiles for cluster analysis. We used Spearman's rank correlation with Average linkage analysis in Cluster 3.0 (PMID: 14871861) and visualized in Java TreeView for the cluster analysis.


When tested by the NCI against a range of human cancer cell lines, it was found that panicein A2 exhibits very little antiproliferative activity at 10 μM – an observation that is at odds with the earlier report that stated panicein A2 exhibits in vitro cytotoxicity against a number of tumour cell lines.The first total synthesis of the unusual aromatic sesquiterpene panicein A This hy2 (5), an example of a cyclised variant of the panicein structure, is a racemic compound that was first isolated in 1994 from Reniera mucosa alongside its non-cyclised isomer 1, and ten other members of the panicein family [2 (5) and D (2) were reported to exhibit in vitro cytotoxicity against four cancer cell lines  with an ED50 of 5 μg/mL [1 (4) was active against P388, A549 and MEL20 cell lines (ED50 = 2.5 μg/mL). The diversity of antiproliferative activities shown by these compounds, and particularly panicein A2 (5), presents them to be attractive synthetic targets.Panicein An family . Panicei2 (5) could be synthesised from the cyclisation and subsequent deprotection of propargyl ether 8. Ether 8 could be formed through the addition of the appropriate phenol to acetylenic alcohol 9, which itself can be derived from aldehyde 10 , giving 10 in 51% yield over two steps  being the double-addition species 19  through a modified Claisen rearrangement [20 in toluene for 48 hours, no desired product 5 was obtained, with a complex mixture of compounds produced  in 80% yield. The NMR data for synthetic panicein A2 (5) matched literature values  exhibited activity against four cancer cell lines with ED50 = 5 μg/mL ≈ 15 μM. To further expand upon these promising results, synthetic panicein A2 (5) was tested by the NCI against 57 human cancer cell lines, through their developmental therapeutics program. Interestingly, at the tested concentration of 10 μM, panicein A2 (5) showed very little activity against most cell lines. The best performance of panicein A2 (5) was against T-47D, a breast cancer cell line, in which it showed a 43% reduction of growth when compared to a control. Two of the cell lines tested by the NCI were the same as those tested against in the original isolation study (A549 and HT29). Our results show that panicein A2 (5) only reduces growth of A549 human lung carcinoma by 14% and had no effect on the HT29 human colon cancer cell line compared to control. These results indicated panicein A2 (5) to have poor antiproliferative activity and the possibility that the originally tested natural material was contaminated with trace amounts of an even more active compound.When isolated, natural panicein A2 (5) has been achieved. This synthesis hinges on key steps involving the addition of phenol 16 to carbonate 18 to provide propargyl ether 8 which was then cyclised through a modified Claisen rearrangement to ultimately give the desired cyclic structure of 5. The correlation of literature values for the isolated natural product and synthetic panicein A2 (5) confirm the structure of the natural product. Of particular note, when synthetic 5 was tested against a broad range of human cancer cell lines, it was found to exhibit very little activity at 10 μM – this observation is at odds with the earlier report that stated 5 exhibits in vitro cytotoxicity against a number of cell lines (ED50 = 5 μg/mL).In conclusion, the first total synthesis of aromatic sesquiterpine panicein AFile 15 and NCI testing results sheet.Experimental procedures, characterisation data of new compounds, NMR comparison tables of natural and synthetic File 21H/13C NMR spectra of synthesised compounds.


Primary mediastinal B-Cell lymphoma (PMBL) is a recently defined entity comprising ~2–10% non-Hodgkin lymphomas (NHL). Unlike most NHL subtypes, PMBL lacks recurrent gene rearrangements to serve as biomarkers or betray target genes. While druggable, late chemotherapeutic complications warrant the search for new targets and models. Well characterized tumor cell lines provide unlimited material to serve as preclinical resources for verifiable analyses directed at the discovery of new biomarkers and pathological targets using high throughput microarray technologies. The same cells may then be used to seek intelligent therapies directed at clinically validated targets. Four cell lines have emerged as potential PMBL models: FARAGE, KARPAS-1106P, MEDB-1 and U-2940. Transcriptionally, PMBL cell lines cluster near c-HL and B-NHL examples showing they are related but separate entities. Here we document genomic alterations therein, by cytogenetics and high density oligonucleotide/SNP microarrays and parse their impact by integrated global expression profiling. PMBL cell lines were distinguished by moderate chromosome rearrangement levels undercutting cHL, while lacking oncogene translocations seen in B-NHL. In total 61 deletions were shared by two or more cell lines, together with 12 amplifications (≥4x) and 72 homozygous regions. Integrated genomic and transcriptional profiling showed deletions to be the most important class of chromosome rearrangement. Lesions were mapped to several loci associated with PMBL, e.g. 2p15 (REL/COMMD1), 9p24 , 16p13 ; plus new or tenuously associated loci: 2p16 (MSH6), 6q23 (TNFAIP3), 9p22 (CDKN2A/B), 20p12 (PTPN1). Discrete homozygous regions sometimes substituted focal deletions accompanied by gene silencing implying a role for epigenetic or mutational inactivation. Genomic amplifications increasing gene expression or gene-activating rearrangements were respectively rare or absent. Our findings highlight biallelic deletions as a major class of chromosomal lesion in PMBL cell lines, while endorsing the latter as preclinical models for hunting and testing new biomarkers and actionable targets. Primary mediastinal B-Cell lymphoma arises in the mediastinum from transformed thymic B-cells and comprises 2–10% NHL. According to microarray profiling, PMBL is distinct from both germinal center and activated diffuse large B-cell lymphomas (DLBCL) bearing the closest pathological resemblance to classical Hodgkin lymphoma (cHL) nodular sclerosing subtype and mediastinal grey zone lymphoma.Although PMBL responds initially to chemotherapy subsequent poor prognostic outcomes warrant the search for new targets and disease models , 2. LikeIn the quest for PMBL biomarkers and pathological targets we have assembled a panel of PMBL cell lines and documented genomic alterations therein using high density arrays offering circa 40–80x improvements over earlier studies. Candidacies of gene targets were evaluated by parallel expression array profiling and reference clinical data. Several new or unfamiliar potential oncogenomic targets were thus identified. Concordance with clinical data thus observed strengthens the validity of PMBL cell lines as useful models and resources.FARAGE was established before 1992 from the lymph node of a 70-year old female at diagnosis of DLBCL sited parasternally . The cloTo eliminate the risk of cross contamination STR DNA profiling was carried out using fluorescent PCR in combination with capillary electrophoresis as described previously . BrieflyCytogenetic analyses were conducted as described previously . Imaginghttp://dgv.tcag.ca/dgv/app/home) for immediate identification of polymorphic CNV.CytoScan High Density Arrays which combine oligonucleotide and SNP probes  were used to detect genomic copy number alterations/gains/losses (CNA/G/L), losses of heterozygosity (LOH) and unbalanced chromosome translocation breakpoints at high resolution. DNA was prepared using the Qiagen Gentra Puregene Kit (Hilden/Germany). Labeling, hybridization and washing were performed using the recommended kits and CytoScan HD arrays according to the manufacturers protocols. Quality control criteria were those set by the manufacturer. Data were subsequently analyzed using the Chromosome Analysis Suite software version 2.0.1.2 (Affymetrix). This allows access to the Database of Genomic Variants (DGV) (www.broadinstitute.org). For creation of heat maps we used the software CLUSTER version 2.11 and TREEVIEW version 1.60 (http://rana.lbl.gov/EisenSoftware.htm). For clustering the algorithm was set to Euclidean distance based on p-values confirmed by multiscale bootstrap resampling [Total RNA (500 ng) were labelled with biotin according to the 3´ IVT Express Kit (Affymetrix). Circa 7.5 μg of biotinylated cDNA were fragmented and placed in a cocktail containing four biotinylated hybridization controls  as recommended by the manufacturer. Samples were hybridized to Affymetrix GeneChip HG-U133 2.0 Plus for 16 h at 45°C. Washing and staining were performed with the fluidics station 450 according to the recommended FS450 protocol. Image analysis was performed on GCS3000 Scanner and GCOS1.2 Software Suite (Affymetrix). Analyses of microarray data were performed using GeneSpring 11.5.1 . Signal intensities (raw data) were log2 transformed and normalized. Comparison datasets were generously provided by Prof. Andreas Rosenwald  or obtained from the BROAD Institute . cDNA was subsequently synthesized from 3 μg RNA by random priming, using Superscript II . qPCR was performed by the 7500 Fast Real-time PCR System , using SsoFast EvaGreen Real Mastermix  and custom designed oligonucleotides . Positive authentication was subsequently provided by cytogenetics (see below).STR profiles of PMBL cell lines are given in Table A in Unsupervised clustering of microarray expression data were displayed as a dendrogram  showing Consensus ISCN karyotypes  of the fFARAGE: 46(41–46)<2n>XX; no consistent abnormality detected. The originator karyotype was: idem +11. Loss of a chromosome 11 homolog is probably attributable to clonal divergence.KARPAS-1106P: 49(45–51)<2n>XXX,+X,i(X)(p10),der(X)dup(X)t, del(2)(p16p22), der(3)t, +9,i(9)(p10),dup(12)(q11q14.1),del(14)(q12q21), del(15)(q11q15),+17, del(18)(q21q22), dup(18)(q12q21),+20,del(20)(q13.1q13.3)x2,add(21)(p13); and that of the originator: 49,XX,der(X)t,i(Xp),del(2)(p11.2p13), der(3)t, +i(9p), ins(q13q13),del(14)(q11.2q13),del(15)(q11q15), der(18)t,-20, del(20)(q13q13)x2. Thus, both karyotypes are closely similar.MEDB-1: 47(41–47)<2n>XY,inv(X),+1,der(1)t,t,+9, der(10)t(q25)(q11),-14,i(21)(q10). The originator karyotype was: 47,XY,inv(X)(p22q13), +der(1)t,+9,-14,-21,i(21q), again closely resembling that observed.U-2940: 45(43–45)X,-X,del(3)(p14p21),del(6)(q13q15),der(7)t, dup(12)(q13q22), der(14)t,t,del(17)(p13); and that of the originator: 45–46,-X,del(3)(p13p21),del(6)(q14q16),del(7)(p11),+i(7)t, dup(12)(q12q21), der(14)del(X)(q21.1q21.3)t,t, again resembling that observed.For representative SKY images see Thus, karyotypes of all four cell lines matched the originators’ data, both confirming their stability in vitro and authenticity suggested by their unique STR profiles  all tested normal, excluding rearrangements at these loci. Thus, although most PMBL cell line karyotypes bore chromosome translocations, none target known common lymphoma breakpoints.Cytogenetically visible deletions at 16p13 hosting SOCS1 were present in 3/4 cell lines: monoallelic in FARAGE, biallelic in Karpas-1106P and U-2940, but absent from MEDB-1 . Only inFISH mapped breakpoints of unbalanced der(3)tp14;p25) present in KARPAS-1106P to ACTR2 at 2p25 and RAF1 at 3p14 . Refinem present Copy number variations/amplifications/deletions (CNV/A/D) and zygosity were investigated with high density combined oligonucleotide SNP arrays. Images depicting each chromosome are shown in Classical CNA which promotes oncogene activity by increasing mRNA levels occurred only sporadically in PMBL cell lines: singular amplifications, moderate in level (3.5–6.5 fold), were detected in three cell lines . Of 12 CFocal deletions abolish gene expression whether acting alone or in concert with inactivating mutations or epigenetic modifications of residual alleles. Focal null deletions were observed in three cell lines: namely, FARAGE (2p16), KARPAS-1106P ; and U-2940  together with 1p12. Of these, three loci bore overlapping null deletions, at 1p12, 9p21 and 16p13. Validation of deletions covering the SOCS1 locus was obtained by FISH for three cell lines, FARAGE, KARPAS-1106P and MEDB-1 , and forParallel microarray expression data  were als1p12: The CD58 locus was targeted by microdeletion, biallelic in KARPAS-1106P, monoallelic in MEDB-1 and U-2940 . Express1q31: The DENN/MADD Domain I (DENND1B) gene which is upregulated in PMBL cell lines is amplified in MEDB-1 only (not shown).2p15: Although genomic array data confirmed the reported CNA in KARPAS-1106P at 2p15, its peak plateau (4n) excluded REL, the mooted target in PMBL . REL exp2p16: Both MSH6 and the adjacent FBXO11 were deleted in FARAGE  and MEDB-1   accompan3q27: Expression of BCL6 and LPP like DLBCL surpassed that in cHL cells placing PMBL closer to the former entity . BCL6 co6q23: Biallelic deletion affecting TNFAIP3/A20  accompan7p22: Focal biallelic deletion of AIMP2 and EIF2AK1  accompan7q31: A complex pattern of deletion and amplification was observed. ZNF277 was downregulated in U-2940, and IMMP2L in MEDB-1 (not shown) which respectively bore focal monoallelic deletions at these loci . In PMBL8q24: A complex CNV pattern was observed around PVT1 and the associated miR cluster while excluding MYC . The cennot shown). In patients as in cell lines CDKN2A was conspicuously silenced, and CDKN2B unevenly so .17q21: GPATCH8 uniquely bore a singleton biallelic deletion in U-2940 which abolished transcription, while the monoallelic deletion in FARAGE accompanied depressed microarray expression therein (not shown).19q13: A biallelic focal deletion was present in U-2940  accompan21q12: A discrete fourfold amplicon of circa 15 Kbp was present in both KARPAS-1106P and MED-B1  which alIn addition to biallelic deletions which ipso facto enforce gene silencing, 49 shared monoallelic deletions (SMD) were recorded . On averLOH are thought to promote cancer by exposing recessive gene mutations or epigenetic silencing modifications among tumor suppressor genes. Alternately, LOH may activate oncogenes by silencing their cognate micro-RNAs. In total 72 shared LOH were recorded the most frequent and widespread alterations present . Most LOBy integrating genomic with parallel transcriptional data sets we both documented genomic alterations in PMBL cell lines and prioritized gene candidates for further assessment. Given the degree to which cell lines portray the emerging oncogenomic picture of PMBL we conclude that they provide suitable resources for pursuing further investigations into this enigmatic neoplasm. Shared impactful changes were shown to feature in two or more cell lines forming patterns of recurrence warranting further study. Although their rearrangement spectrum partially overlaps neighboring entities cHL and DLBCL, PMBL cell lines stand apart bearing far fewer rearrangements than the former, while lacking the recurrent B-cell oncogene translocations of the latter. These evidences plus its snug transcriptional niche close to both neigboring entities show that the PMBL cell lines fill a unique oncogenomic niche consistent with their attribution to PMBL.The likely import of deletions in PMBL was first bestowed by low density array studies , 26. WhiAmong validated candidates, SOCS1 deletion/mutation in KARPAS-1106P and MEDB-1 has been already described where it delays JAK2 degradation to allow pSTAT6 stockpiling , 30. OurIn 21% DLBCL cases the immunosurveillance marker CD58 (LFA3) is inactivated , as reflFBXO11 at 2p16 forms a complex which targets BCL6 for ubiquitylation and proteasomal degradation in DLBCL . BiallelCentromeric of FBXO11 lies REL at 2p15 which though highly expressed in PMBL cell lines  falls ouTNFAIP3 inactivating mutations occur widely in DLBCL, cHL and in PMBL patients and have been reported in KARPAS-1106P , 43. MutAlthough deletions affecting 9p21 are widespread in aggressive leukemia and have been described in DLBCL, genes at this locus emerge relatively unperturbed in PMBL . Here liOur data also show that the posited genomic upregulation of JAK2 which outlies the CNA peak also warrants further study. Although JAK2 is constitutively phosphorylated in MEDB-1 at least , “smokinHigh density oligonucleotide/SNP arrays remove some bias inherent in BAC arrays while offering improved resolution, parallel LOH data, and—thanks now to the continually updated Database of Genomic Variants—the ability to sift out natural polymorphic CNV. Integrating genomic and transcriptional microarray data allows prioritizing of gene candidates targeted by genomic rearrangements at a first pass level. By delineating ever shorter regions of interest, high density arrays simplify candidate ranking. Finding little evidence that chromosomal translocations impact cancer gene alterations, our findings pinpoint bilateral microdeletions as the predominant class of impactful genomic lesion in PMBL cell lines. Thus our findings strengthen the case for applying high density arrays to clinical samples. And while LOH was in a few cases also correlated with gene silencing, its likely transcriptional—hence oncogenic—consequence remains doubtful. Parallel studies addressing the impact and role of gene mutations in PMBL cell lines are nearing completion in this lab, while those addressing the role of epigenetic modifications, such as DNA methylation, in gene regulation in PMBL are underway.This study combined high density oligonucleotide/SNP arrays with cytogenetics to profile the genomic features of PMBL cell lines revealing subtle genomic changes therein, notably short genomic deletions. By integrating genomic lesions with global cell line and patient expression data we shortlisted potentially impactful gene targets therein, comprising known, suspected and hitherto inconspicuous candidates. The current investigation endorsed the validity of this panel to serve as an oncogenomic resource and scaffold in pursuit of novel biomarkers and actionable gene targets in PMBL and evaluate “intelligent” therapies directed against specific lesions in cells bearing them.S1 FigAbbreviations: AML, acute myeloid leukemia; DLBCL, diffuse large B-cell lymphoma; erythro-megakaryocytic leukemia; ITL, immortalized T-cell; PMBL, primary mediastinal B-cell lymphoma; TCL, T-cell lymphoma.A: Transcriptional profiling of PMBL and other hematopoietic cell lines was used to construct a cluster diagram–(updated from ref. 10)—of mRNA expression. The AU value (printed red) gives the "approximately unbiased" p-value, which is calculated by multiscale bootstrap resampling. The bootstrap probability value is less stringent than AU value when testing significance. Clusters (edges) with high AU values are strongly correlated. Results are shown as a dendrogram, showing different expression profiles as early dividing branches. B: PCA plot of microRNA expression. Note discrete clustering of both mRNA and miR expression showing that PMBL cell lines occupy a unique niche apart from other hematopoietic entities. (TIF)Click here for additional data file.S2 FigShows absence of product yielded for ACTR2-RAF1 fusion suggested by genomic breakpoints and reported recently . Amplifi(TIF)Click here for additional data file.S3 FigSee legend to (PDF)Click here for additional data file.S4 FigShows global microarray expression for select genes in PMBL patients compared to cHL. Data extracted from ref .(PDF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


Limited research exists regarding the most aggressive forms of hepatoblastoma. Cell lines of the rare subtypes of hepatoblastoma with poor prognosis are not only difficult to attain but also challenging to characterize histologically. A community-driven approach to educating parents and families, regarding the need for donated tissue, is necessary for scientists to have access to resources for murine models and drug discovery. Herein, we describe the currently available resources, existing gaps in research, and the path to move forward for uniform cure of hepatoblastoma. Hepatoblastoma is the most common primary liver tumor diagnosed in childhood , with apThe disease predominantly occurs in young children, from birth to 5 years of age . The hisClinically, the empirically driven advancements in postoperative chemotherapy and surgery, including the multidisciplinary approach set forth through the Pretreatment Extent of Disease guidelines , has impScientists and clinicians are now seeking non-chemotherapeutic treatments for patients with unresectable or metastatic tumor – treatments that directly target the molecular underpinnings of hepatoblastoma progression. For example, clinical trials regarding cixutumumab and pazopanib, monoclonal antibodies, and alisertib, a kinase inhibitor, have all been completed or are actively being investigated in phase 2 clinical trails for the treatment of refractory hepatoblastoma .In order to find other targeted therapies, researchers need hepatoblastoma tissues and cell lines. There is an absence of diversity in hepatoblastoma cell lines for scientists and clinicians to use to better understand the disease. In this paper, we describe the need for more cell lines and murine models to advance the discovery of therapeutic targets for the more aggressive subtypes of hepatoblastoma.via PubMed was conducted to obtain information on research with unique hepatoblastoma cell lines and murine models. First, the authors found the number of distinct hepatoblastoma cell lines published in the literature. The search terms used were “‘hepatoblastoma’ [All Fields] and ‘cell line’ [MeSH Terms].” The search provided approximately 450 publications, all reviewed by the authors of this paper. Any publication that had a focus on hepatoblastoma was mentioned in Table An extensive literature review of hepatoblastoma Manuscripts that had differing characterizations of certain cell lines were included in the “Unsure Hepatoblastoma” portion of Table via PubMed to find murine models of hepatoblastoma. The search terms used were “hepatoblastoma murine models.” This provided approximately 50 publications that the authors reviewed. All publications that studied murine models of hepatoblastoma were included in Table Next, the authors reviewed literature via PubMed and found no manuscript publications. The authors then searched the European Journal of Cancer using the term “hepatoblastoma xenograft” and found a published abstract using patient-derived xenografts, which is included in Table Finally, the authors searched for patient-derived xenografts Data regarding histological characterization and experimental murine models from only a few hepatoblastoma cell lines exist. These cell lines tend to have a favorable histology, leading to an underrepresentation of the high-risk subtypes .Fifteen hepatoblastoma cell lines are described in current literature Table . AdditioMost confirmed cell lines are of the mixed histology subtype. However, there are no cell lines of the small cell undifferentiated subtype, which carries the worst prognosis .Only one chemically induced murine model of hepatoblastoma has been reported Table . AlthougTen unique cell line-derived subcutaneous xenografts and three cell line-derived orthotopic murine models of human hepatoblastoma exist Table . These min vitro .Grassroots communication and interaction among family members, caregivers, and others affected by a particular diagnosis has significantly influenced progress in some areas of pediatric cancer research. As an example, interaction among families affected by diffuse intrinsic pontine glioma (DIPG) in an online discussion group is considered by some to be the first step in raising a tide turning awareness in that community. The result was a promising therapeutic drug, panobinostat, for treatment .In summary, the greatest potential for the development of targeted therapy for aggressive forms of hepatoblastoma will come when scientists have access to hepatoblastoma cells lines and tissues with histological subtype diversity Figure .RR, KS, RH, MSB, MB, and LH: contributed significantly to the acquisition, intellectual content, and final approval and are in agreement with all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


KRAS that may confer radioresistance. We hypothesized that mutant KRAS can regulate glutamine metabolism genes in NSCLC and maintain tumor redox balance through transamination reactions that generate cytosolic NADPH via malic enzyme 1 (ME1), which may contribute to radioresistance.Advanced non-small cell lung cancer (NSCLC) is an aggressive tumor that is treated with a combination of chemotherapy and radiation if the patient is not a candidate for surgery. Predictive biomarkers for response to radiotherapy are lacking in this patient population, making it a non-tailored therapy regimen with unknown outcome. Twenty to 30 % of NSCLC harbor an activating mutation in KRASG12D driven NSCLC and patient data was analyzed from multiple publicly accessible databases including TCGA, CCLE, NCBI GEO and Project Achilles. ME1 expression was found to be mutant KRAS associated in both a NSCLC mouse model and human NSCLC cancer cell lines. Perturbing glutamine metabolism sensitized mutant KRAS, but not wild-type KRAS NSCLC cell lines to radiation treatment. NSCLC survival analysis revealed that patients with elevated ME1 and GOT1 expression had significantly worse outcomes after radiotherapy, but this was not seen after chemotherapy alone.A doxycycline-inducible mouse model of KRAS driven glutamine metabolism genes, specifically ME1 and GOT1 reactions, may be a predictive marker and potential therapeutic target for radiotherapy in NSCLC.The online version of this article (doi:10.1186/s13014-015-0457-x) contains supplementary material, which is available to authorized users. KRAS . In. InKRAS KRAS NSCLC cell lines from the Cancer Cell Line Encyclopedia (CCLE) revealed that genes involved in glutamine dependent redox balance (ME1 and GOT1) were significantly upregulated in mutant KRAS cell lines with normalized enrichment scores (NES) >1.48  of wild-type vs mutant alance ME and GOT1KRASG12D; p53−/− transgenic mouse model of NSCLC to examine mRNA expression in the KRAS induced (“ON”) and extinguished (“OFF”) states  was induced vs 48 h extinction with doxycycline withdrawal (n = 4 mice)  of a tetracycline operator-regulated Tet-op-tes Fig. . When fepression . Within ce) Fig. . We founME1 and GOT1 in 11 mutant and 11 wild-type KRAS NSCLC cell lines and found both genes to be significantly upregulated in the mutant cell lines  media for 16 h, then exposed to ionizing radiation and allowed to form colonies for 7 days. Short-term Gln deprivation did not significantly alter clonogenic survival on its own, but did sensitize HCC44 and not H522 cells to radiation, at normally sub-lethal doses  NSCLC patients who were treated with IR  . –12. in vapy Fig. , c. Furters Fig. , e. Last ~7 % 1/1 of the tKRAS associated glutamine metabolism genes, GOT1 and ME1, as potential radioresistance biomarkers in NSCLC. Our study revealed that elevated expression of GOT1 or ME1 is a highly predictive biomarker in radiation treatment, but not chemotherapeutic, outcomes. Additionally, ~93 % of patients with a complete response to IR treatment harbored wild-type KRAS in their tumors. To explain these observations, we hypothesize that KRAS-reprogrammed glutamine flux through GOT1 and ME1 is critical to maintain cytosolic NADPH levels for redox balance and lipid synthesis in NSCLC. In the face of ROS stress, as observed with IR treatment, NADPH is preferentially used to maintain reduced glutathione and thioredoxin 1 to protect cells from ROS damage [KRAS may reprogram NSCLC glutamine metabolism similar to that observed in pancreatic cancer to maintain redox balance, thus providing an oncogene driven mechanism of radioresistance. While there are currently no known specific inhibitors of ME1 or GOT1, targeting upstream glutamine utilization via glutaminase 1  (http://www.broadinstitute.org/ccle) [https://tcga-data.nci.nih.gov/tcga/tcgaCancerDetails.jsp?diseaseType=LUAD&diseaseName=Lung adenocarcinoma) [http://www.broadinstitute.org/achilles) [GSEA of mutant vs wild-type rg/ccle) . Transgercinoma) . Level 2chilles) .n = 73) was conducted using the Kaplan-Meier Plotter webtool (kmplot.com) [Survival analysis in radiation treated NSCLC patients (lot.com) . Brieflyhttp://cancergenome.nih.gov/), no patients were approached for this study. No consent and no ethical approval were required to utilize this database.All human data is sourced through The Cancer Genome Atlas , allowed to grow for 7 days, washed with PBS and stained with crystal violet solution. Colonies with >50 normal appearing cells were counted and percent survival calculated and graphed with dose.5 cells per plate and transfected with either control siRNA or siRNA against ME1 for 48 h followed by clonogenic survival assay.For siRNA transfection, cells were plated in 10 cm plates at 2 × 10