In HIV-1-infected patients a long lasting CD4+ cell decline influences the host-EBV balance and thereby increases the risk for EBV related malignancies. In spite of a world-wide access to combination antiretroviral therapy (cART) there are still a considerable number of HIV-1-infected patients who will develop severe immunodeficiency. These undiagnosed HIV-1 infected patients, so called late testers, demonstrate an increased lymphoma risk, compared to patients diagnosed early. Consecutive individual screening for EBV DNA-load in late testers might be a useful predictor of emerging EBV-malignancy.in vitro and further analyzed.Patient biopsies and ascites were analyzed morphologically, by immuncyto-histochemistry and in-situ hybridization. Viral DNA and RNA load were quantified by PCR. Cell lines from primary tumor and from ascites, were established 6/L. The lymphoma was CD45-negative and weakly CD22- and CD30-positive. The patient had a history of Kaposi sarcoma and HHV-8 seropositivity. The lymphoma biopsies, and three cell lines derived on different occasions from the tumor cell effusion, were all EBV-positive but HHV-8 negative.We here report on a case of EBV-positive lymphoma in an AIDS patient, first presenting with pleural effusion and ascites and was thus initially considered a primary effusion lymphoma (PEL) but was later diagnosed as a plasmablastic lymphoma (PBL). The patient had responded to cART with undetectable HIV-RNA and increased CD4 cell count one year prior to lymphoma presentation. At the time of lymphoma diagnosis the HIV-RNA values were <50 RNA-copies per mL blood (undetectable) and the CD4-positive cell count 170 ×10A noticeable EBV-DNA load decline was observed during the remission of the lymphoma following CHOP-therapy. The EBV-DNA load increased dramatically at the time of recurrence.EBV DNA load might be useful in monitoring the effect of lymphoma treatment as well as in estimating the risk of EBV-associated lymphoma in HIV-1 infected patients with pronounced immunosuppression. EBV infects the vast majority of humans and resides latently in B-cells. The virus carries genes that can induce and maintain mature B cell growth. In immunocompromised post-transplant and HIV-infected patients the virus may cause lymphoproliferative or malignant diseases. Although EBV negative lymphoid malignancies have been described in HIV-infected persons EBV has a strong association with a wide range of B-cell lymphomas in HIV-carriers including Burkitt’s lymphoma (BL), Hodgkin lymphoma and Diffuse Large B-cell lymphomas .Plasmablastic lymphoma (PBL) is a rare aggressive subtype of non-Hodgkin lymphoma (NHL) most commonly localized to mucosal tissues such as the oral cavity, but may also be manifest in other organs such as liver, breast or bone . MorpholFurthermore, PBL is CD45-negative, while PEL is CD45-positive. Both PBL and PEL display markers associated with plasma cell differentiation such as CD38 and CD138 and usually express cytoplasmic immunoglobulin and may be variably positive for CD30. Although most cases of PBL occur in AIDS-patients with profound immunodeficiency, HIV-1 negative cases have also been reported [In the era of modern combination antiretroviral therapy (cART) there has been an impressive reduction of AIDS-related opportunistic infections and lymphomas. However, HIV-1 patients still suffer an increased risk for NHL  and the 6/L. In a malignancy assessment the same year endoscopic biopsies were taken from the gut mucosa. By routine histopathological analysis the biopsies showed signs of chronic inflammation and macrophage infiltration, but no lymphoma. Bone marrow aspiration was also performed with May-Grunewald stained smears showing immature cells of the myeloid series and many lymphoid cells (44%), including cells of lymphoplasmocytoid/plasmacell type. Staining for kappa and lambda light chains gave no evidence for clonal B-cell proliferation. The findings were suggested to represent a polyclonal B-cell hyperplasia without suspicion of lymphoma.When the patient was diagnosed with HIV-1 the CD4+ cell count was 170 × 10Oral hairy leukoplakia was diagnosed early the year after HIV-diagnosis and the same year the patient developed hemolytic anaemia Figure . The ana6/L, and CMV-retinitis was diagnosed and treated with altering foscarnet and ganciclovir. During the fourth year HIV-RNA values increased to 65 000/mL prompting a change in the anti-retroviral treatment to AZT and didanosine. Second half of fourth year cART was introduced . After cART introduction, including a protease inhibitor, HIV-RNA was <500/mL, and remained undetectable during the following two years. The patients CD4 cell count went from 30 × 106/L in second half of year two after HIV-1 diagnosis to 190 × 106/L after introduction of cART year four. The suppressive CMV-treatment was withdrawn the same year (year 4).In December the second year antiretroviral treatment with azidothymidine (AZT) was initiated, and shortly thereafter a conjunctival Kaposi lesion was removed. In February the following year , and one month later ascites. CT scan revealed a mass in the gastric wall and pleural effusion. An initial cytological analysis of the effusion suggested a primary effusion lymphoma (PEL). A biopsy from the tumor in the gastric wall confirmed the presence of an EBV-positive Non-Hodgkin lymphoma with immunoblastic features considered to be a manifestation of PEL.After diagnosis of the lymphoma cytostatic treatment with cyclophosphamide, hydroxydaunorubicin (Doxorubicin), vincristine (Oncovin), prednisolone (CHOP), and intrathecal methotrexate was initiated and the ascites disappeared within four days. At the time of lymphoma treatment Rituximab was not yet approved and was therefore not a treatment option. The patient continued with cART and suppressive toxoplasmosis treatment. The CHOP treatment was repeated every 3rd week and the patient completed six treatment cycles in total. Before every CHOP treatment blood was drawn for EBV-DNA analysis. EBV-load was also measured on three occasions the month after completing CHOP treatment. The remission of the tumor mass of the gastric wall was confirmed with a CT-scan and the patient did clinically well. Then, one month after the final CHOP course, ascites reappeared together with pleural effusion. Reintroduced cytostatic treatment now only had marginal effect on the lymphoma. Pleural effusions were evacuated twice. The patient died four and a half years after HIV-1 diagnosis due to progression of the lymphoma.At autopsy lymphoma manifestations were observed in spleen, liver, lymphoid organs and lymphomatous dissemination to pleura and peritoneal cavity. A small cerebral mass in globus pallidus was also found, but this was not further analyzed to rule out lymphomatous spread to the central nervous system lymphoma or a lesion related to the patient’s earlier diagnosed cerebral toxoplasmosis.Retrospective in situ hybridization of the patient’s early Kaposi manifestation did show EBER-expression in some (probably lymphatic) cells.When the patient presented with a gastric tumor and ascitic and pleural effusions, cytological examination of the ascitic fluid showed a population of dispersed large blastic cells with a high frequency of mitoses and partly vacuolated dark blue cytoplasm. The tumor cells were morphologically highly reminiscent of blastic lymphoid cells, with features of immunoblastic plasmablastic differentiation Figure . By immuA trans-abdominal core biopsy was subsequently obtained from the extra nodal tumor in the gastric wall, which was prepared for flow cytometry and histopathology and molecular analysis. The routine hematoxylin-eosin (H-E) staining showed an infiltration of a large cell malignant lymphoma with immunoblastic/plasmablastic features, but without any mucosal component or lymph node structures in the specimen Figure . The morAdditional analysis was performed on autopsy material. Immunohistochemical analysis on sections was positive for CD43 and CD138  than SPIBL-II and III (one fragment at 87–92 kD and one at 108 kD), which both have the same clonal origin.The clinical features of this patient’s lymphoma manifestations were at the time of diagnosis consistent with primary effusion lymphoma (PEL), with ascitic and pleural effusions. Gene expression profile analysis of PEL has suggested a plasmablastic derivation in common with PBL . NeverthHIV-associated PBL is considered as a very aggressive lymphoma, with a poor prognosis also in the era of cART .As far as could be analyzed on the autopsy material this lymphoma had histopathological and phenotypical features that were similar at first diagnosis and after recurrence. At relapse the lymphoma was spread both to extra-nodal sites and reappearing in effusions.The CD4 expression on the PBL cells was unexpected, but has been seen in other cases, as has recently been reviewed . MoreoveThe cell lines derived from the biopsies taken before and after recurrence were distinctly different. Cell lines from the early lymphoma had a lymphoblastoid cell line feature, i.e. similar to immunoblastic lymphoma. The cell lines derived from pleural effusion at recurrence were BL like, but did not have the t translocation typical for BL. They expressed EBV-latency program I/II, which altogether would be compatible with the plasmablastic phenotype, also suggested by other markers on the cell lines. The cell lines were also different from reported HHV-8-positive PEL lines, which regularly do not express any immunoglobulin .From clonal analysis of immunoglobulin-rearrangement we can conclude that the cell lines seem to share one marker of rearrangement of the heavy chain locus, while another marker is seen in addition to the BL like/PBL-cell lines. This might indicate that they represent clonal progression from the original lymphoma precursor cell, although it is more likely that they are derived from different clones. SPIBL-I could also be a spontaneous lymphoblastoid cell line derived from latently EBV infected cells not related to the tumor, although its origin from the ascitic effusion would be compatible with a tumor origin.At the time of progression the HIV-RNA was non-detectable and CD4+ cell count had increased, although not to an acceptable level. A persevering decreased CD4+ cell count can be reflected in the lack of EBV control . A long Our finding of an EBV-DNA decline and persistence of low EBV-DNA loads clearly reflects the cytostatic treatment effect on the lymphoma. The subsequent, pronounced EBV-DNA increase preceding the lymphoma relapse suggests a value of EBV monitoring in treatment as well as follow up of EBV-positive lymphomas. This indicates the use of EBV-DNA load as a marker for recurrence as well as a treatment effect marker. The use of EBV-DNA load has also been clearly shown in the assessment of risk for EBV-positive PTLD in transplanted patients -25.In HIV-1-infected patients a long lasting decline of CD4+ cells will disrupt the host-EBV balance and increase the risk for EBV related malignancy. In spite of the worldwide access to cART and awareness of HIV-1 there are still a considerable number of undiagnosed HIV-1-infected individuals, who will have developed severe immune deficiency at the time of HIV diagnosis. When initiating cART treatment at the time of HIV-diagnosis in this patient group , repeated analysis of EBV- could be of value in estimating the lymphoma risk.In this rare PBL with effusion our finding of EBV-DNA decline and persistence of low EBV-DNA load reflects the positive effect of cytostatic treatment on the lymphoma. This finding together with a pronounced EBV-DNA increase preceding the lymphoma relapse demonstrates the value of EBV monitoring in treatment of EBV-positive lymphomas in AIDS-patients. The result indicates the usefulness of EBV-DNA load as a marker for recurrence and treatment effects. EBV DNA-load might also be useful as an indicator of lymphoma risk in HIV-infected patients presenting late in HIV-1 infection with severe immunodeficiency.6/L, antiretroviral treatment was initiated and in year three the patient began with cART. In the middle of year four the patient was diagnosed with a lymphoma. The lymphoma went into remission after cytostatic treatment, but recurred three months later with massive pleural effusion. The patient died in middle of year five. Biopsies were taken from the gut the first year, four years prior to the lymphoma diagnosis and from the developed lymphoma on three occasions in year five.A 36 year old homosexual man was diagnosed with HIV-1 in 1993. Two years later a Kaposi conjunctival lesion was removed . For immunocytochemistry an alkaline phosphatase chromogenic substrate was used, whereas for immunohistochemistry a peroxidase-based detection system was employed according to the manual . Pre-treatment protocols for antigen demasking on paraffin sections were used according to standard procedure for each antibody.Flow cytometry was performed with a Becton-Dickinson analyser (Becton-Dickinson).Tissue biopsy materials, aspiration from ascitic and pleural effusions, were collected upon surgical excision or aspiration from the patient, stored in sterile 0.9% NaCl and were set up within 4 hours for tissue culture. Biopsy material was minced with scissors and forceps and passed through a metal wire mesh 3–4 times. The cell suspension thus generated was set up in 12-well microplates with RPMI 1640 and antibiotics. The cultures were followed regularly by microscopical examination and fed by replacing some half the culture medium once every 4–7 days in the beginning. Cells from the first biopsy occasion were transferred to microplates with irradiated fibroblast feeder layer. After one-two weeks clumps or loose aggregates of cells were detected and they were explanted in five mL Falcon plastic tubes on feeder layers after 2–3 months, and subsequently upon expansion of the cells to 25 mL Falcon bottles . The second and third attempts to establish cell lines (SPIBL II and III) from the relapsed pleura effusions were very different and cells grew out much faster than on the first attempt, already 1–2 weeks after in vitro explanation. The established cell lines were used for characterization and also stored away by freezing.The cell lines were analyzed by standard light microscopy.Chromosomes in the cell lines were analyzed by making metaphase plates and stained for Giemsa-banding, according to Zech et al. .Cellular DNA was analyzed using a PCR method .EBV-PCR from blood analysis and statistics were performed as previously reported by Friis et al. . EBV wasEBV: Epstein-Barr virus; cART: Combined anti-retroviral treatment; HIV-1: Human immunodeficiency virus 1; HHV-8: Human herpes virus number 8; PEL: Primary effusion lymphoma; PBL: Plasma blastic lymphoma; SPIBL: Spontaneous immunoblastoid cell line.No commercial relationship or potential conflict of interest related to the submission exists. No non-financial competing interests exist.AF participated in the design of the study, carried out EBV laboratory work and wrote manuscript. BÅ participated in the design of the study, recruited patient and wrote manuscript. KG participated in the design and coordination of the study, recruited patient and wrote manuscript. AA carried out sample preparation and cell line related laboratory work. IE participated in the design of the study, wrote manuscript. BC made pathology evaluations and wrote manuscript. JZZ carried out HHV-8 laboratory work. All authors read and approved the final manuscript.


Triticum aestivum carrying such genes and control lines. We hypothesized that competitive performance of GM lines would be reduced due to enhanced transgene expression under pathogen levels typically encountered in the field. The transgenes pm3b from wheat (resistance against powdery mildew Blumeria graminis) or chitinase and glucanase genes from barley  were introduced with the ubiquitin promoter from maize (pm3b and chitinase genes) or the actin promoter from rice (glucanase gene). Phytometers of 15 transgenic and non-transgenic wheat lines were transplanted as seedlings into plots sown with the same 15 lines as competitive environments and subject to two soil nutrient levels. Pm3b lines had reduced mildew incidence compared with control lines. Chitinase and chitinase/glucanase lines showed the same high resistance to mildew as their control in low-nutrient treatment and slightly lower mildew rates than the control in high-nutrient environment. Pm3b lines were weaker competitors than control lines. This resulted in reduced yield and seed number. The Pm3b line with the highest transgene expression had 53.2% lower yield than the control whereas the Pm3b line which segregated in resistance and had higher mildew rates showed only minor costs under competition. The line expressing both chitinase and glucanase genes also showed reduced yield and seed number under competition compared with its control. Our results suggest that single transgenes conferring constitutive resistance to pathogens can have ecological costs and can weaken plant competitiveness even in the presence of the pathogen. The magnitude of these costs appears related to the degree of expression of the transgenes.Genetically modified (GM) plants offer an ideal model system to study the influence of single genes that confer constitutive resistance to pathogens on the ecological behaviour of plants. We used phytometers to study competitive interactions between GM lines of spring wheat  Advances in biotechnology have allowed the introduction of single genes against fungal pathogens into plants Resistance to a pathogen might reduce plant fitness when the pathogen is absent from the environment. Such constitutive resistance is often associated with costs resulting from the allocation of resources to unnecessary defense in a pathogen-free environment, making these resources unavailable for other fitness-relevant processes Using genetically modified (GM) cereals as a model system allowed us to control the genetic background of experimental lines and to ensure that the GM lines differed only in one resistance gene from non-GM control lines. With this approach we could avoid the problem that resistant plants might differ in multiple resistance and other genes from control plants, which often hampers interpretation in studies of natural populations or in conventional agricultural crops The desired outcome of introducing genes that confer resistance into plants is that the benefits of resistance may outweigh the potential costs in the presence of the pathogen. Under disease pressure, the introduced trait should lead to better pathogen defense and thus increased fitness of the plants carrying the transgene compared with those lacking it, perhaps allowing transgenic plants or their offspring to become invasive in natural habitats environmentsplants  to environmental conditions We used a phytometer approach Triticum aestivum L. variety Bobwhite SH 98 26, hence abbreviated Bobwhite, transformed with the wheat pm3b gene that confers resistance to powdery mildew Blumeria graminis f.sp. tritici (DC.) Speer chitinase and glucanase from barley The spring wheat Apart from providing information about the effects on plant × environment interactions of single pathogen-resistance genes, the assessment of the competitiveness of transgenic and conventional wheat in crop environments contributes to understanding the potential risks associated with offspring of GM plants potentially occurring and competing with conventional wheat in subsequently sown fields. Furthermore, the potential for enhanced performance of phytometers when grown with another line (“away environment”) instead of its own (“home environment”) would suggest a positive effect of growing wheat in line mixtures, an effect abundantly found in biodiversity experiments www.NRP59.ch). Other research projects within the Consortium have studied agronomic properties of the wheat lines in common agricultural trials, gene × abiotic environment interactions in the glasshouse and in the field The work presented here is part of a joint project of several research groups called “The Wheat Consortium” within the framework of the Swiss National Research Program 59 “Benefits and risks of the deliberate release of genetically modified plants” (Here we asked the following questions: (1) Do the introduced transgenes improve resistance to mildew and do they affect the performance of the phytometers grown under competition (main effects of transgenes)? (2) How do the nutrient and the competitive environments affect resistance to mildew and phytometer competitive performance (main effects of environments)? (3) Do the differences between transgenic and control lines vary across nutrient and competitive environments ? (4) Do transgenic and control lines behave differently if planted into their own rather than into different lines as competitive environments (home vs. away contrast of transgene × environment interactions)? We found that transgenic constitutive resistance to a fungal pathogen can affect plant × environment interactions and reduce the competitiveness of the GM plants which show strong transgene overexpression.T. aestivum is a predominantly self-pollinating species with hexaploid genome and growing season from early spring to late summer (in Switzerland). Bobwhite and Frisal were chosen because these varieties are known for high transformation efficiency and regeneration frequency We used four transgenic lines derived from the Mexican spring wheat variety Bobwhite and two transgenic lines derived from the Swiss variety Frisal in our experiment. Spring wheat Pm3b#1–4) and their non-transgenic control sister lines (Sb#1–4) were produced by biolistic transformation in four different transformation events. Pm3b#1–3 lines carried a single copy of the transgene pm3b, and Pm3b#4 line carried one full-length and one non-functional truncated copy pm3b gene or other variants of the pm3 gene pm3b gene confers race-specific resistance to powdery mildew and was cloned from the hexaploid wheat landrace Chul The transgenic lines of Bobwhite  pm3b gene quantified in the leaf samples from the field in 2008 by reverse transcription, quantitative real-time polymerase chain reaction (RT-qPCR) were similar among Pm3b#1, Pm3b#3 and Pm3b#4 lines, while the line Pm3b#2 showed around five times higher expression levels Pm3b#3 line, where some plants showed high resistance and others were susceptible to mildew Pm3b#2 and in a highly resistant subset of Pm3b#3. We hypothesized that these unintended effects were related to a very high transgene expression The performance in monoculture and the transgene expression of the lines chitinase gene (line A9 Chi) or both a chitinase and a β-1,3-glucanase gene (line A13 Chi/Glu) Chi/Glu and A9 Chi. No transgene silencing occurred in these lines . In the absence of sister lines that had undergone the same tissue culture as the transgenic Frisal lines, we used ordinary non-transgenic Frisal plants as the control line. Here we present the results of the first field experiment carried out with these plants. All GM lines used were produced as model plants for the National Research Program 59 and were not intended for agricultural commercialization.The GM lines derived from the variety Frisal expressed either a barley seed within varieties with differences between varieties, and thus to verify whether the characteristics of the GM plants fall within the range of natural variation between conventional varieties of wheat, the criterion of the test of equivalence required by the European Food Safety Authority for risk assessment of GM plants In addition to the 11 lines already mentioned, four further wheat lines were used: ordinary non-transgenic Bobwhite plants that had not passed through tissue culture and the three commercial non-transgenic Swiss varieties: Casana, Fiorina and Toronit. The latter were used as reference “out-groups” to compare differences caused by the transgenes −2 as “Ammonsalpeter 27.5”, Lonza, Visp, Switzerland). The natural field soil provided plants with phosphorous, potassium and magnesium .The field experiment took place in 2008 at a research station in Zurich-Reckenholz, Switzerland. The 15 wheat lines were sown in 60 plots of 7×1.08 m each, in a randomized complete block design with four replicate blocks. Each plot represented one of the 15 wheat competitive environments for the phytometers. The two edge subplots of 1×1.08 m in each plot were used for a split-plot treatment, fertilizer application vs. control. Fertilizer was applied twice: when the plants had reached phenological stage 11 on the “Zadoks” scale −1 Bromoxynil, 120 g L−1 Ioxynil, 100 g L−1 Fluroxypyrmetilheptilester; Omya Agro AG, Safenwil, Switzerland) at the beginning of May. Mildew infection occurred naturally. As a subset of these plots (environments but not the phytometers) had provided the plant material for one of our previous publications Pm3b and sister lines) and traits were assessed.In each 1×1.08 m subplot, 400 wheat seeds were sown in six rows with a distance of 18 cm between the rows using an Oyjord plot drill system . Five seedlings per subplot were randomly chosen and marked shortly after germination for later assessment of mildew incidence in the sown competitive environments. All plots were sprayed with the herbicide cocktail Concert SX  and Starane super  were germinated in a climate-controlled glasshouse  at the Institute of Evolutionary Biology and Environmental Studies, University of Zurich, Switzerland. When the seedlings reached phenological stage 11–12 on the Zadoks scale Thirty phytometer seedlings representing the 15 wheat lines were introduced into each 1×1.08 m subplot . Before We recorded plant height and phenological stage Pm3b lines vs. 4 Sb lines, A9 Chi and A13 Chi/Glu vs. Frisal), (2) and (3) comparing GM and control lines pairwise . All data were log-transformed to fulfill ANOVA assumptions of normality and homoscedasticity. The binary mildew incidence data were analyzed using multiple logistic regression with mixed-model analysis of deviance Data were analyzed with classical mixed-model analysis of variance (ANOVA) using the statistical software GenStat . The treatment model consisted of the factorially-crossed phytometer lines and competitive environments  and fertilizer application. The error model consisted of phytometer plants nested within subplots, subplots nested within plots and plots nested within blocks. The terms of the treatment model were tested against the appropriate terms of the error model: competitive environment varied among plots, fertilizer application among subplots and phytometer line within subplots . ResiduaFirst we analyzed the originally measured variables to identify the differences in phytometer performance and the effects of competitive environment and fertilizer application. Then we analyzed the relative performance values (see previous section) to compare the competitive ability of phytometer lines independently of their different performance in “pure stands” (i.e. phytometer plant in its own competitive environment). For this analysis we calculated the log-ratio of away/home as a dependent variable see e.g. . For eacIn June 2008, 1093 out of 3600 phytometer plants were damaged by vandals. These plants were excluded from the analysis of the traits measured after the damage had happened. ANOVA showed that the damage by vandalism occurred randomly across the phytometer lines and did not interfere with the effects of the factors of interest.pm3b gene showed the desired decrease in mildew incidence (Pm3b lines vs. Sb lines phytometer contrast: P<0.001); this decrease was up to five-fold compared with control lines . The four Pm3b lines, however, differed significantly from one another in mildew incidence (4 Pm3b lines phytometer contrast: P = 0.01). Overall, line Pm3b#2 had the lowest and line Pm3b#3 had the highest mildew scores with, respectively, 6% and 14% of the phytometers infected. The four control Sb lines only marginally differed from each other in mildew incidence (4 Sb lines phytometer contrast: P = 0.059) and were highly susceptible to the pathogen (up to 62% of the plants infected).Each Pm3b and Sb wheat lines done in the field at subplot level Pm3b lines showed higher resistance to the pathogen than the sister lines, Pm3b#2 line being the most resistant to powdery mildew among the four GM lines.The results obtained with phytometers were similar to the results of other mildew assessments of the same P<0.001).The lines derived from the susceptible Mexican variety Bobwhite had a 14-fold increased mildew incidence compared with the lines derived from the Swiss wheat variety Frisal , however, mildew incidence was also low in the Frisal control line. Mildew incidence in Frisal plants never exceeded 7% of all phytometers in any line x fertilizer treatment combination .The lines A9 bination . In the P<0.001). The competitive environment also affected mildew incidence (main competitive environment effect: P = 0.001). Higher mildew rates were observed for the phytometers introduced into mildew-susceptible wheat environments (Pm3b plots (Pm3b vs. Sb lines competitive environment contrast: P<0.001). Mildew incidence did not differ between the phytometers grown in Frisal transgenic and control competitive environments.Application of fertilizer led to a twofold increase in mildew incidence of phytometers . Competitive environment also affected the magnitude of the difference in mildew incidence between Pm3b and Sb lines (interaction Competitive environment × Pm3b lines vs. Sb lines: P = 0.024). This difference was 3.4 times stronger in mildew-susceptible Sb than in mildew-resistant Pm3b competitive environments.The difference in mildew incidence between Pm3b lines on average developed 45.4% less seeds, 39.4% lower yield and 4.8% lower vegetative mass, and had a more advanced phenological stage and plant height  than control Sb lines  ; the four control Sb lines in contrast differed only in vegetative mass (4 Sb lines phytometer contrast: P = 0.018) with lower values observed for the lines Sb#2 and Sb#3 than for the other two lines  . In addiwo lines  and S3.Pm3b#2, which exhibited the highest transgene expression Pm3b GM lines: yield was reduced by 53.2%, seed number by 48.1% and vegetative mass by 27.3% compared with the corresponding control Sb#2 . This line also had 8.8% more advanced phenological stage and 13.2% taller plants than the control line . The three other Pm3b lines differed less from their controls. The line Pm3b#1 had 12.7% reduced yield, 17.5% reduced seed number and 10.2% reduced vegetative mass compared with its sister line Sb#1 . Pm3b#4 showed 7.9% lower yield and 18.4% lower seed number (Pm3b#4 vs. Sb#4 phytometer contrast: P<0.001 for yield and seed number) and had slightly advanced phenological stage compared with its sister line Sb#4 . Line Pm3b#3, which had higher mildew incidence than the three other Pm3b lines (Pm3b#3 vs. Sb#3 phytometer contrast: P = 0.001 for seed number).The GM line 3b lines , differePm3b and Sb lines exceeded the variation among the three conventional wheat varieties Casana, Toronit and Fiorina for the traits yield, seed number and vegetative mass several times .The transformed lines derived from the Swiss wheat variety Frisal had an advanced phenological stage and a 1.5-fold increased plant height compared with the lines derived from the Mexican variety Bobwhite. Bobwhite lines, on average, had a 1.3-fold increased seed number per plant and yield, 1.2-fold increased spike number and 1.5-fold increased vegetative mass compared with Frisal lines .Line A9 P<0.001 for all the traits) causing a 2.4-fold increase in yield, 2.3-fold increase in seed number and vegetative mass, 1.4-fold increase in spike number, an advance in phenological stage and a 1.2-fold increase in plant height of the phytometers.Nutrient addition enhanced plant growth and development . For these traits, the differences between Pm3b and Sb competitive environments exceeded the variation among the three conventional-wheat-variety environments . The phytometers planted in the different Frisal environments  only varied in phenological stage, which was delayed in phytometers grown in the transgenic A9 Chi compared with those grown in Frisal environment .Phytometers which had Frisal lines as competitive environments had delayed phenological development compared with those planted into Bobwhite lines as competitive environments .Nutrient addition did not significantly change the magnitude of the differences in performance between Pm3b lines and control Sb lines . The difference between Pm3b and Sb phytometers in yield increased 2.1-fold, in seed number 1.7-fold and in vegetative mass 5-fold when the plants were grown in Frisal as compared to Bobwhite environments.The competitive environments Frisal vs. Bobwhite significantly affected the differences in yield, seed number and vegetative mass between P = 0.009, P = 0.032 and P<0.001 for log-ratios of yield, seed number and vegetative mass) and plant height and phenological stage were lower in “away” than in “home” environments . This was indicative of higher performance (biomass) due to reduced light competition (lower height) of phytometers in “away” environments.Overall, yield, seed number and vegetative mass were higher . However, on average, relative plant height and phenological stage were 4% higher for the Pm3b lines than for the Sb lines .On average, Bobwhite control lines and conventional Swiss varieties performed better in away than in home environments whereas the opposite was the case for GM lines : Pm3b liPm3b lines differed significantly in their relative yield, spike number, seed number, plant height and vegetative mass (4 Pm3b lines phytometer contrast: P<0.001) . This confirms the results on absolute performance of Pm3b#3 line under competition: this line had only minor differences compared with its sister control line.Not all lines contributed to the same degree to the mentioned average differences between GM and non-GM lines. The four P<0.001)  and S5. Chi and A13 Chi/Glu had slightly advanced phenological development and plant height in away compared to home environments, whereas the Frisal control line was more phenologically advanced and taller in home than in away environments . The line A13 Chi/Glu and Frisal variety had increased vegetative mass in away as compared to home environments, whereas A9 Chi line showed no such effect .Frisal transgenic lines A9 P = 0.014, P = 0.001, P = 0.007, P = 0.021 for log-ratios of yield, seed number, vegetative mass and phenological stage, respectively), indicating that line mixtures may be less beneficial under high than under low soil nutrient conditions.Nutrient addition reduced the overall positive away/home log-ratios of yield, seed number, vegetative mass and phenological stage  indicate that the pm3b gene is also constitutively expressed with its indigenous promoter. The control Mexican wheat variety Bobwhite and the null-segregants used as sister control lines carried no indigenous pm genes. Therefore we suggest that the differences in performance between the Pm3b and the control lines could be explained by the high expression of the pm3b gene in transgenic lines. However, because we could not compare the performance of our transgenic Bobwhite lines with that of the landrace Chul, we cannot exclude the possibility that even with the original promoter the pm3b might have reduced plant performance under the prevailing pathogen pressure.Increased mildew resistance, however, did not lead to enhanced growth and competitive performance of the tested GM lines in the presence of the pathogen. On the contrary, the plants with These costs of resistance also indicate that, at least under the environmental conditions encountered in our field experiment, the mildew-resistant GM lines do not have a higher chance than conventional lines to establish and persist as volunteers in wheat habitats. There is a discussion in the literature if the addition of a single gene can cause a crop to become weedy Pm3b lines with different expression levels, we found significant differences between the four transgenic lines in their performance and interactions with the different competitive environments. Thus, line Pm3b#2, which showed the highest resistance to powdery mildew, was the weakest competitor, re-enforcing the view that transgene-caused, high mildew resistance was negatively correlated with plant performance. When the average yields are plotted against the average mildew incidence for all Frisal and Bobwhite lines  in our study.The data obtained at individual plant level in the phytometer experiment supported the results of our previous glasshouse and plot-level field assessments of the same Chi/Glu, which expressed both chitinase and glucanase transgenes, had lower yield and seed number than the Frisal control line. In accordance with this observation, line A13 Chi/Glu also showed an increased resistance compared with the control line in fertilized subplots. Again it appears that additional investment into pathogen resistance, which was already elevated in the Frisal control line, was costly for the Frisal GM line expressing two transgenes. That performance was not reduced in the Frisal line expressing only one transgene (A9 Chi) suggests that the degree of defense matters for the costs of defense. We conclude that a high constitutive level of mildew resistance has negative effects on the performance of GM wheat plants and thus reduces their potential to persist in conventional agricultural fields. It could be that lower levels of intrinsic resistance to pathogens might produce better-performing GM plants. From a risk perspective, however, such plants would have to be evaluated again in a range of biotic and abiotic environments in similar experiments as the one presented here to test their or their offspring's potential to successfully compete with non-GM plants.Line A13 Solidago canadensisOur second question was how variation in the abiotic (fertilization) and biotic environment (competition with other wheat lines) may influence resistance to mildew and the performance of phytometer plants. Nutrient addition enhanced powdery mildew incidence in both transgenic and conventional wheat lines. Similar effects were reported in previous studies with non-transgenic plants, where the severity of mildew infection was shown to be related to the nitrogen supply of the host Pm3b lines as competitors outperformed those planted into competitive environments of Sb lines. This is in accordance with the results of the analysis of the main effects of transgenes (see previous section). Phytometers which had Frisal variety as a competitive environment generally had weaker performance than those in Bobwhite environments. The congruence between phytometer-line and competitive environment-line effects, i.e. high-performing phytometer lines also providing highly competitive environments thus in turn reducing phytometer performance, indicates that phytometers do provide realistic measures of competitive ability.Fertilization enhanced plant growth and reproduction in all the investigated wheat lines. In addition, the performance of the phytometer plants was strongly influenced by the type of competitive environment. Phytometers planted with transgenic The third question asked whether transgenic wheat lines responded to variations in nutrient and competitive environments in the same way as did conventional lines. The difference in mildew incidence between GM lines and control increased with the addition of nutrients. A similar effect has been previously described in non-transgenic plants, where the increased severity of infection due to fertilization was more pronounced in susceptible than in resistant crop varieties and therefore the magnitude of the difference between these varieties increased with nutrient addition Pm3b lines of variety Bobwhite than for other lines. Our findings indicate that a single gene that confers constitutive resistance to a pathogen might strongly affect genotype × environment interactions if expressed at a high level, making ecological costs of resistance apparent even in the presence of the pathogen. The fact that the differences between GM and control lines in pathogen level and plant performance vary depending on the environment points to the importance of testing transgenic plants under a set of biotic and abiotic environments in realistic field conditions Significant transgene × competitive environment interactions were observed for the majority of fitness-related traits and reflected more sensitive responses to competition for transgenic 2 in the field. An advantage of the phytometer approach is the possibility to incorporate several biotic and abiotic factors that might affect the performance and competitive ability of test plants simultaneously into a single and comprehensive experimental setting Using this approach we could assess competitive interactions and the response to fertilizer treatments in 15 different transgenic and conventional wheat lines simultaneously on a relatively small area of less than 130 mPm3b lines of variety Bobwhite, however, showed lower relative values (performance in “away” as compared to “home” environments) than the corresponding control lines for four out of six fitness-related traits. Only the line with partial gene silencing, Pm3b#3, showed no such costs under competition with the other lines. Because the GM line Pm3b#2 suffered most in mixtures, it appears that this line paid a particularly high fitness costs for its elevated mildew resistance under competition. This observation supports the recent findings that competition might increase the magnitude of the costs of resistance Pm3b#2, appeared to be especially sensitive to inter-line competition, whereas the differences between this line and its sister control line became smaller when the competitor was represented by its own genotype. Interestingly, the reduced performance and fecundity under competition with the other wheat lines (relative performance) was also observed in the Pm3b#4 line  rather than into different lines as competitive environments (away). Most of the phytometer plants benefited if their neighbors belonged to a different line (mixture effect). This is consistent with findings in biodiversity experiments b#4 line  which isTransgenic plants with unintended phenotypes, including low fecundity, often arise during molecular plant breeding et al. showed that herbicide-tolerant transgenic lines of rape showed no evidence to be more successful or more invasive than their conventional counterparts in the absence of herbicide treatment and even showed weaker invasive potential in some aspects, such as in seed survival after burial presence of the pathogen against which the GM lines had increased resistance. Very likely we would have observed even higher costs in our study if the pathogen would have been excluded in our field trial.Our results confirm these precedents and demonstrate that a transgene increasing plant resistance to a pathogen and constitutively expressed at a high level may reduce rather than increase a plant's competitive ability and thus lower its probability to persist outside its own field. An early study of Crawley Apart from these findings, nutrient addition negatively affected the ability of plants to coexist in the mixtures. This supports the theory that fertilization increases competition between genotypes or species for scarce resources and in particular light In conclusion, this study shows that a single gene conferring resistance against a particular fungal pathogen can have large and negative effects on plant performance under realistic field conditions even if these conditions include the presence of the pathogen. We interpret these large costs in resistant plants as a consequence of altered gene regulation, in particular enhanced gene expression level, which was here achieved with a strong promoter introduced with the gene that confers resistance. This indicates that altered regulation in a single gene may strongly affect plant fitness and the way the plant interacts with the environment, in particular changing a plant's competitive ability.Figure S1Design of the phytometer experiment.(PDF)Click here for additional data file.Figure S2The structure of orthogonal contrasts used in the extended ANOVA models.(PDF)Click here for additional data file.Table S1Analysis of deviance table showing the effects of fertilizer, competitive environment, differences between GM and non-GM lines and their interactions on mildew incidence.(PDF)Click here for additional data file.Table S2ANOVA table showing the effects of fertilizer, competitive environment, differences between GM and non-GM lines and their interactions on three yield characteristics.(PDF)Click here for additional data file.Table S3ANOVA table showing the effects of fertilizer, competitive environment, differences between GM and non-GM lines and their interactions on phenological stage, plant height and vegetative mass.(PDF)Click here for additional data file.Table S4ANOVA table showing the effects of fertilizer, competitive environment, differences between GM and non-GM lines and their interactions on three relative yield characteristics.(PDF)Click here for additional data file.Table S5ANOVA table showing the effects of fertilizer, competitive environment, differences between GM and non-GM lines and their interactions on relative phenological stage, plant height and vegetative mass.(PDF)Click here for additional data file.


Salermide is a potent inhibitor of SIRT1 and SIRT2 and can induce tumor-specific cell death in selected human cell lines. In this study we investigated salermide’s apoptotic effect in a wide range of other human cancer cell lines and its antiproliferative potential in combination with cisplatin.The NADSeven different cancer cell lines  were treated with salermide (1 µM – 0.1 nM) for 24, 48, and 72 hours and assessed for cell viability. Three cell lines  were selected for combination therapy with salermide and cisplatin (30 µM). In order to characterize salermide’s proapoptotic pathway SIRT1, SIRT2, pAKt, p53, acetyl-p53 and Nampt (nicotinamide phosphoribosyltransferase) were determined in SKOV-3 and Jurkat cells by Western blotting.0-G1 cell cycle arrest. SIRT1 and SIRT2 protein were down-regulated after 48 h and 72 h. This was accompanied by a down-regulation of pAKT, p53 and Nampt. Acetyl-p53 levels were not consistent across cell types. Cisplatin exerted synergistic effects with salermide in all cell lines and reduced cell viability up to 50%.Salermide yielded greater dose-dependent apoptotic effects in Jurkat, SKOV-3 and N87 cells than in the other cell lines, with most potent effect after 48 h of incubation. The anti-proliferative activity was associated with a GSalermide-induced apoptosis is cell line-dependent and more effective in slow-proliferating (SKOV-3) and hematologic (Jurkat) cancer cells. The synergism with cisplatin implies a potentiating effect of this sirtuin inhibitor as add-on in clinical cancer therapy.


The aim of this study was to provide further information about triggering cell death and suggestion of methods for the elimination of unwanted cells such as tumor cells.Scorpion venom toxicity is of major concern due to its influence on human activities and public health. We investigated the Bothutus saulcyi, does not show cytotoxic effect on any of the used cell lines. Odontobuthus doriae, however, has resulted a dose dependent cytotoxic effect with maximum at 1 ug/mL on 1321N1 glioma like cell line. Then the cytotoxic venom of O. doriae was fractionated using Sephadex G50 gel chromatography. The toxic fractions on mouse used to Cytotoxicity assay on 1321 N1 cell line and data demonstrated that, the fraction F3 showed a dose dependent Cytotoxicity assay. Further studies to explode the mode of action of these venoms are recommended and purification of the toxic fraction should be done.The cytotoxicity and apoptosis induced by effect of scorpion venoms on five established eukaryotic cell lines are analyzed on different human cell lines. All cultured cell lines were incubated with varying doses of scorpion venom for 24 h at 37°C. Control culture was treated with an equal amount of SFM. The percentage of cell survival was measured using the 3--2,5-diphenyltetrazolium (MTT) colorimetric assay. Our data demonstrated that  Odontobuthus and Buthus  as modified by BioRad Inc., using bovine gamma globulin as a standard. Enzyme specific activities and all other analyses were based on these protein concentrations. Standard curve was drawn using bovine serum albumin (BSA) for any new plate or assay. Cell lines: Five different human cell lines have been used in this investigation. The HEK293 cell line that is derived from human embryo kidney and is transformed with sheared human Ad5 DNA which is sensitive to human adenoviruses and adenovirus DNA, OV2008 (Ovarian cell line), 1321N1 , HEPG2 , and A549 (established from an explanted lung tumor which was removed from a 58-year-old Caucasian man in 1972). All cells were propagated in EMEM  or DMEM (1321N1 and A549) medium  supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine, and the antibiotics penicillin  and streptomycin (100 mg/L). The cell lines were incubated in flasks as monolayers and cultured at 37°C with 5% CO2 in a fully humidified atmosphere. Cytotoxicity assay: For the purpose of examining the effects of scorpion venoms on cell growth, a colorimetric 3--2,5-diphenyl tetrazolium bromide (MTT) assay was used. This assay is based on the cellular conversion of a tetrazolium salt (MTT) into a formazan product that is easily detected using a 96-well plate reader. Venoms were initially diluted in PBS and then serial dilutions were prepared using serum free cell culture medium in 96-well microtiter plates (Costar). Cells (1.5 × 104 to 0.5 × 105 cells well) were added into wells containing various concentrations of venoms , and grown for 24 h. At 24 h, cell culture medium in each well was replaced with 200 μL of medium containing 0.5 mg/mL MTT, followed by incubation at 37°C for 3 h. DMSO was added into each well and the optical density at 570 as the base and 650 nm as the test was measured by a microplate reader (Bio-Rad 550). In each experiment, six wells were used, and experiments were repeated three times. 50 ug/mL of Cisplatin was used as a cytotoxic agent for control group.O. doriae, was dissolved in 10 mL of 0.1 M ammonium acetate in water (pH 8.6). The solution was centrifuged for 30 min in 5000 rpm and the non soluble part removed and fractionation carried out with clarified sample in Sephadex G50 column (210 × 3.5 cm) in room temperature. Flow rate was 1 mL/min. Absorbance was measured at 280 nm and the content of tubes of each curve was mixed. The fractions were dialyzed in 1500 D cut off for 24 h and lyophilized at -50°C. Protein content determined as described above. Lethality tests and LD50 determination in mice: The mice  used in this research were treated in compliance with the US Public Health Service policy on human care and the use of animals. The mouse lethality of various protein fractions was measured using Muench and Reed method after IV injections to mice.Fractionation Procedure: 500 mg of the crude venom of Toxic fractions were determined and used for Cytotoxicity assay on 1321N1 cell line. Statistical Analaysis: GraphPad Prism 3.02 version software was used for statistical analysis. One-way ANOVA and Newman-Keuls Multiple Comparison Test were used for analyzing of data.The dried scorpion whole body has been widely used in traditional Chinese medicine based on the Chinese herbalism ‘combat poison with poison’. Even at the present time, it is still used as a drug to treat neurological symptoms such as painful headaches, convulsion, seizures, swellings and paralysis .O. doriae and B. saulcyi venoms on human cell lines. The antiproliferative and cytotoxic effect of venoms were supported by cell count using MTT assay. At longer time points, 48 h, the antiproliferative effect was reduced (unpublished data), which might be due to venom denaturation by the presence of temperature in aqueous solutions. That is why a 24 h exposure was used to determine the cytotoxicity of venom in most of experiments. The present study was conducted to evaluate the antiproliferative activity of The Bradford Protein concentration determination showed that absorption of the sample was 0.36. It means that the concentration of the sample was 9 ug/mL. this data was used as standard for the next steps .B. saulcyi venom, a kind of resistance to the cytotoxicity effect is apparent for the HEK cell line (Although HEK cell line is very much sensitive to the cytotoxic agents as is shown for cisplatin in our study, but seems to be very much resistant to either venoms. In case of ell line .B. saulcyi venom. This cell line was not sensitive to O. doriae either. There is no significant reduction in HEK cell count in the presence of different doses of O. doriae venom ( In fact, except for a minor and not significant decrease in cell number after exposure to 0.25 ug/mL of venom, no significantly reduction in cell population is shown after exposure to ae venom .in-vitro exposure to these venoms. The OV2008 cell line was not sensitive to either O. doriae or B. saulcyi venoms, with no significant difference between control group and groups that exposed to different doses of venom (Three other cell lines, OV2008, A549 and HepG2, look very much the same after 24 h of venom  and 5. O. doriae or B. saulcyi after in-vitro exposure (A549 cell line has neither shown any sensitivity to exposure  and 7. Texposure  and 9 inB. saulcyi venom (O. doriae. Among the different doses tested at different cell lines 1 ug/mL venom of O. doria at 24 h was found to produce a marked inhibition on the 1321N1 proliferation with a p value of < 0.001 (The 1321N1 cell line did not show sensitivity to yi venom  but it w < 0.001 .O. doriae has only decreased the absorbance of MTT dye on 1321N1 cell line. Based on Cytotoxicity results that showed O. doriae crude venom was toxic on 1321N1 cell line, the venom was fractionated for determining of the toxic fraction. Fractionation with Sephadex G50 gel chromatography, isolated six fractions of O. doriae crude venom. (The hallmark of carcinogenesis is uncontrolled cellular growth and proliferation. In normal cells, cell proliferation and DNA replication is monitored by cell cycle check points and apoptosis. We found that from five different cell lines in our Cytotoxicity assays, the venom of e venom. + channels (+ channel blockers, and chlorotoxin, a 36 amino acid peptide, that specifically binds to small conductance Cl channels (Lethality test showed that fractions F3, F4 and F6 were toxic in mouse.  these frchannels , short-cchannels .O. doriae that are active on voltage-gated Na channels and potassium channels have been studied widely (in-vitro cytotoxicity of O. doriae venom on 1321N1 glioma cells among all of above studied cell lines, is due to the presence of the plenty of vital ion channels in this cell line, and the blocking effects of the scorpion venom components on these channels. Further studies to investigate on these mechanistically hypothesis are recommended. Up to now, toxins isolated from d widely , 19 in gd widely -22. Blocd widely -29. Gliod widely -36. Thes


Clinical utility and applications of these MGPs will need to be carefully examined with relevant clinical outcome measurements and constraints in practical use.Previous studies have reported conflicting assessments of the ability of cell line-derived multi-gene predictors (MGPs) to forecast patient clinical outcomes in cancer patients, thereby warranting an investigation into their suitability for this task. Here, 42 breast cancer cell lines were evaluated by chemoresponse tests after treatment with either TFAC or FEC, two widely used standard combination chemotherapies for breast cancer. We used two different training cell line sets and two independent prediction methods, superPC and COXEN, to develop cell line-based MGPs, which were then validated in five patient cohorts treated with these chemotherapies. This evaluation yielded high prediction performances by these MGPs, regardless of the training set, chemotherapy, or prediction method. The MGPs were also able to predict patient clinical outcomes for the subgroup of estrogen receptor (ER)-negative patients, which has proven difficult in the past. These results demonstrated a potential of using an  Breast cancer remains a significant cause of mortality in women with nearly 40,000 deaths in the U.S. during 2010 alone in vitro drug response of NCI60 cell lines to predict patient chemotherapy response were not successful Recently, researchers have attempted to overcome the limitations of patient sample-derived MGPs by using cancer cell lines to develop MGPs, with varying degrees of success in vitro assay, the selection and number of cell lines, the platform and quality of array measurements, and the statistical method employed, may contribute to this discrepancy. To address these questions, two different sets of breast cancer cell lines were exposed to two combination chemotherapies–TFAC  and FEC –and assayed by an in vitro chemoresponse test. We also independently developed our MGPs using two prediction methods, supervised principal component regression (superPC) and CO-eXpression ExtrapolatioN (COXEN), developed by the groups at Precision Therapeutics, Inc. and the University of Virginia, respectively. We subsequently validated these MGPs in five clinical trials with patient gene expression profiling data and full clinical annotation of chemotherapy treatment and outcome. The goal of this systematic investigation was to objectively evaluate the effectiveness of cell line-derived MGPs as tools to guide clinical decisions in the application of standard chemotherapies.These conflicting data on the utility of cell line-derived MGPs highlights the need for further and complete evaluation, including for those MGPs developed from breast cancer cell lines. Many factors, including the precision of the Table S1) were obtained from either ATCC  or DSMZ . RPMI 1640 medium  containing 10% FBS  was used to maintain all of the cell lines at 37°C in 5% CO2. Before conducting in vitro chemoresponse tests, each cell line was trypsinized and seeded into 384-well microtiter plates  after reaching roughly 80% confluence.Forty-two breast cancer cell lines , Ten serial dilutions, in triplicate, of the TFAC combination of paclitaxel , 5-fluorouracil , doxorubicin , and pre-activated cyclophosphamide , or the FEC combination of 5-fluorouracil , epirubicin , and pre-activated cyclophosphamide  plus media controls were prepared in 10% RPMI 1640 medium before being used to treat each cell line. Both combination treatments were composed of equal volumes of each drug at each dose. The cells were incubated at 37°C in 5% COhttp://www.ncbi.nlm.nih.gov/geo/, accession number GSE12777, labeled here as “Hoeflich” http://www.ebi.ac.uk/, accession number E-TABM-157, labeled here as “Neve” http://rmaexpress.bmbolstad.com/) using default settings, except that the probe-level model analysis method was used to summarize probe values. Before further analyses, the probe-level intensities were log2-transformed. Probe sets that had low levels of variation (interquartile range <0.5) or low expression values (median<log2 [100]) were non-specifically filtered out across all cell lines. The expression values were then standardized to a mean of zero and a standard deviation of one for each cell line.The gene expression omnibus database  under the programming environment R 2.11.1(http://www.r-project.org/). In short, the association between the cell line chemoresponse-derived AUD scores and the expression values for each probe set was analyzed by univariate linear regression analysis. In a linear regression model, the first principal component was chosen to predict the result of patient chemotherapy, as measured by pCR. A lower prediction score corresponded with greater chemotherapy sensitivity and therefore a higher likelihood of achieving pCR. To investigate the impact of the number of predictor genes, this parameter was varied from 50 to 1000.Supervised principal components (superPC) regression was used to develop the MGPs for TFAC and FEC chemotherapies invitro chemoresponse data was used for TFAC and FEC combination treatments of the 42 breast cancer cell lines to obtain initial candidate expression biomarkers (probe sets) that were the most predictive of the cell lines’ chemosensitivities to each drug combination. Specifically, initial candidate biomarkers differentially expressed between chemo-sensitive and –resistant cell lines were identified from the breast cancer cell lines by two-sample t-test. In addition, biomarkers highly associated with in vitro chemosensitivity were identified by evaluating correlation coefficients between drug sensitivity of each cell lines and gene expression data, both with a false-discovery rate (FDR) <0.05. These chemosensitivity biomarkers were then triaged based on the COXEN coefficient which represents the degree of concordance of expression regulation between the breast cancer cell lines and a cohort of breast cancer patients. The mathematical derivation of COXEN coefficient is based on the so called “correlation of correlations”, which first calculates the expression correlations within each set on the same set of genes of interest for both sets and then evaluates gene-by-gene correlation between the two correlation matrices of the two sets. For the derivation of COXEN coefficients in this study, a gene expression dataset compiled from 251 breast cancer patients The COXEN method was also used to develop MGPs, as previously described Using the final COXEN biomarkers for each drug combination, MGPs were developed by applying within-gene standardization and a cross-validated principal component regression analysis to each cell line training set to avoid any potential statistical over-fitting in large screening molecular-based prediction modeling. The biomarkers were then sequentially involved in regression model training by the order of strength of chemosensitivity association. Several competing, high-performance models with different numbers of candidate biomarkers were obtained from the training sets. These competing models were then evaluated and compared by utilizing two of the five clinical trials Table 2.For the development of estrogen receptor (ER)-specific prediction models, we divided the cell lines into ER+ and ER– groups based on ESR1 mRNA expression levels, as described elsewhere The chemoresponse of each cell line to TFAC and FEC, represented by AUD scores, can be seen graphically in S5). For MGPs developed from both the Neve and Hoeflich training sets, the performance is stable for numbers of genes, in the range 100–700. In this work, we selected the top 150 genes whose expression values are most correlated with the in vitro chemosensitivity as gene signatures to predict patient chemotherapy response.Using the superPC method, we have developed MGPs for both TFAC and FEC treatments , as well as two drugs that are of the same mechanism of action (A and E). However, the MGPs developed by the Neve and Hoeflich training sets have a relatively small overlap of their biomarkers This may be because these two training sets were measured at different sites and using different platforms (HG-U133 vs HG-U133 plus 2.0). To assess the reproducibility of array data, we calculated the correlation of gene expression based on the 28 cell lines which are common to both training sets, and obtained a correlation of 0.77 for the set of genes that are common in HG-U133 and HG-U133 plus 2.0. The superPC method selects genes based on the association of each gene with drug response; therefore even slight changes in training dataset may lead to a complete different set of genes selected for prediction. Nevertheless, functional analysis indicates that most of these genes are from the same gene functional networks, including cell death, the cell cycle, cellular development, small molecule biochemistry, molecular transport, cellular growth and proliferation, cellular assembly and organization, and cellular function and maintenance. Moreover the scores of predictors generated from Neve and Hoeflich datasets are consistent for all five validation datasets, with the correlation of predictors generated from two training sets all greater than 0.86.S9). For the MGPs based on the Neve training set, 47 and 162 COXEN biomarkers were identified for TFAC and FEC, respectively, of which 14 biomarkers were in common. For the MGPs based on the Hoeflich training set, 124 and 20 biomarkers were selected for each TFAC and FEC, and 17 of 20 biomarkers for FEC were also included in those for TFAC. The gene functions of TFAC COXEN biomarkers were primarily from the cell cycle, cellular growth and proliferation, hematological system development, cellular compromise, and drug metabolism and function. Of these, six genes  have been reported to be directly relevant to cancer mechanisms, such as mammary tumor progression (P = 0.046) Using the COXEN method, we have also developed MGPs for both TFAC and FEC (−6) for all cases, which included different training sets, treatments, and test sets  and Hoeflich (bottom row) training sets. The two prediction methods provided highly correlated scores for both TFAC and FEC chemotherapies with Spearman correlation coefficients between 0.75 and 0.91  analysis to evaluate the overall predictability of our MGPs Figure 4We also evaluated superPC and COXEN MGPs by AUC for ER+ and ER– patients separately. For ER+ patients, both the superPC and COXEN models achieved good prediction results for all but the MAQC-validation cohort Table 3.S17).  The development of ER-specific predictors was similar as before, except that ER– or ER+ predictors were developed separately, based on 22 ER– or 19 ER+ cell lines instead of all 41 cell lines . This may provide a means to efficiently discover and evaluate effective therapeutic options for these aggressive subtypes of breast cancer, which have been limited due to the relatively small proportions of breast cancer patients exhibiting these subtypes. This strategy also allows one to discover and test biomarkers for various single chemotherapy agents and their combinations, including unusual combinations that have occasionally or never been used in clinics.Strategies using in vitro-based MGPs, caution is needed in drawing conclusions from this finding, which should be restricted to the chemotherapies investigated in this study. We believe that one of the key issues in using cell lines to develop MGPs is whether the surrogate cell line system can accurately approximate patient clinical outcomes. Gene expression patterns have been found to vary widely between cancer cell lines and patients, possibly diminishing the suitability of cell lines as patient sample surrogates in vitro system cannot fully mimic a tumor’s in vivo environment or emulate complex in vivo drug metabolism, and cancer cell lines that can proliferate in artificial in vitro environments might represent only a subset of in vivo tumor cells in vitro and in vivo environments. Also, while individual genes are highly variable, MGPs aggregating information from multiple genes have the effect of mitigating this variability. A majority of biomarkers from different prediction methods for each chemotherapy treatment shared the same gene networks and functions, such as cellular growth and proliferation, cell morphology, and cell death, despite the relatively small number of overlapping genes (5 biomarkers for TFAC and 2 biomarkers for FEC). As recently reported, we believe there also exist a relatively large number of biomarkers that can effectively stratify tumors with contrasting chemosensitivity Mixed results have been reported in some previous studies of cell line-derived MGPs ER– and ER+ breast cancers have been widely recognized as having heterogeneous gene expression patterns, different mutations, distinct alterations in DNA copy number, and many other differences Table S1Summary of chemosensitivity of 27 breast cancer cell lines to FEC and TFAC, and the information of gene expression measured by Neve and Hoeflich.(DOC)Click here for additional data file.Table S2MGP-TFAC developed from the Neve training set by the superPC method.(DOC)Click here for additional data file.Table S3MGP-FEC developed from the Neve training set by the superPC method.(DOC)Click here for additional data file.Table S4MGP-TFAC developed from the Hoeflich training set by the superPC method.(DOC)Click here for additional data file.Table S5MGP-FEC developed from the Hoeflich training set by the superPC method.(DOC)Click here for additional data file.Table S6MGP-TFAC developed from the Neve training set by the COXEN method.(DOC)Click here for additional data file.Table S7MGP-FEC developed from the Neve training set by the COXEN method.(DOC)Click here for additional data file.Table S8MGP-TFAC developed from the Hoeflich training set by the COXEN method.(DOC)Click here for additional data file.Table S9MGP-FEC developed from the Hoeflich training set by the COXEN method.(DOC)Click here for additional data file.Table S10MGP-TFAC developed from the ER positive Neve training set by the COXEN method.(DOC)Click here for additional data file.Table S11MGP-TFAC developed from the ER negative Neve training set by the COXEN method.(DOC)Click here for additional data file.Table S12MGP-TFAC developed from the ER positive Hoeflich training set by the COXEN method.(DOC)Click here for additional data file.Table S13MGP-TFAC developed from the ER negative Hoeflich training set by the COXEN method.(DOC)Click here for additional data file.Table S14MGP-FEC developed from the ER positive Neve training set by the COXEN method.(DOC)Click here for additional data file.Table S15MGP-FEC developed from the ER negative Neve training set by the COXEN method.(DOC)Click here for additional data file.Table S16MGP-FEC developed from the ER positive Hoeflich training set by the COXEN method.(DOC)Click here for additional data file.Table S17MGP-FEC developed from the ER negative Hoeflich training set by the COXEN method.(DOC)Click here for additional data file.


In the current study, four lung cancer cell lines harboring wild-type epidermal growth factor receptor, comprising two squamous and two non-squamous cell lines, were used. The IC10 concentrations of three platinum-based regimens were combined with radiation treatment. Cells were irradiated at 0, 2, 4, 6 and 8 Gy using a 137Cs irradiator concurrently or sequentially. Surviving fractions (SFs) were plotted as a function of the radiation dose. In A549 cells, only the docetaxel (Doc) and carboplatin (Carbo) combination showed a significant radiosensitizing effect with CCRT treatment. For the other three cell lines, no difference was identified in the SFs between CCRT and SCRT. An in vitro method of comparing CCRT with SCRT was established using lung cancer cell lines. Overall, no significant difference was detected in the radiosensitizing effect of the two treatment schedules, with the exception of the A549 cell lines treated with Doc/Carbo.Current practice guidelines recommend the combination of chemotherapy and thoracic radiation for locally advanced non-small cell lung cancer (NSCLC). Previous meta-analyses have shown that concurrent chemoradiation (CCRT) may be superior to sequential chemoradiation (SCRT). However, few previous  Lung cancer is the most common cause of cancer-related mortality worldwide . Non-smain vitro data concerning these two treatment schedules are available. The present study conducted in vitro chemoradiation using NSCLC cell lines to detect differences in chemoradiosensitivity according to the treatment schedule used.Compared with clinical data, little 2 in RPMI 1640 with glutamine  containing 5% fetal bovine serum .Four NSCLC cell lines, A549 (adenocarcinoma), H1299 (large cell neuroendocrine carcinoma), HCC15 [squamous cell carcinoma (SQCC)] and H157 (SQCC) were obtained from the Hamon Cancer Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center . These cell lines were maintained at 37°C and 5% CO10 values of these three platinum-based regimens were determined using colony formation assays. The results are presented as the IC10 values of Gem, Pem and Doc in each combination. The IC10 values of Cis and Carbo were then calculated using the drug ratio based on the doses used in the clinic.The following three platinum-based regimens were used: Gemcitabine (Gem)/cisplatin (Cis), pemetrexed (Pem)/Cis and docetaxel (Doc)/carboplatin (Carbo). For chemotherapy combined with radiation, the ICin vitro chemoradiation treatment scheme. For CCRT, the cells were seeded in triplicate six-well plates containing RPMI 1640 plus 10% FBS at various densities commensurate with the dose of radiation. These cells were treated with chemotherapy at the IC10 and with radiotherapy on the same day. For SCRT, the cells were plated on 100-mm dishes and treated with chemotherapy at the IC10 on day 1 (D1). To terminate drug exposure, the medium was removed and the dishes were then washed twice with medium on D4. The cells were allowed to grow for seven days and then irradiated on D11 following replating on six-well plates.137Cs irradiator . Colonies containing >50 cells were stained with crystal violet and manually counted using a microscope. The percentage cell viability of the irradiated samples [shown as plating efficiency (PE)] was calculated as the fraction of cell viability relative to 100% viability of the untreated samples and plotted as a function of the radiation dose. Surviving fractions (SFs) were plotted as a function of the radiation dose. Cell survival curves were generated using the following multitarget, single-hit cell survival equation:The cells were irradiated at various doses  using a 0 indicates the dose required to reduce the SF to 37% and n indicates the total number of targets at 0 Gy. The radiation sensitivity was expressed as the SF at 2 Gy (SF2).S indicates the SF at dose D, while D10 values for each doublet. To these concentrations, radiation was then added concurrently or sequentially. In the H1299, HCC15 and H157 cells, no differences were identified between CCRT and SCRT . Das et al identified a subset of naturally occurring EGFR mutations that lack a critical radioprotective function of EGFR (in vitro studies of combination modalities.The four cell lines used in the current study harbored wild-type epidermal growth factor receptor ( of EGFR . In the in vitro method of comparing CCRT with SCRT using lung cancer cell lines. Overall, no significant difference was detected in the radiosensitizing effect of the two treatment schedules, with the exception of the A549 cell line treated with Doc/Carbo. The underlying mechanism should be elucidated.In conclusion, the present study established an


In an effort to improve product yield of mammalian cell lines, we have previously demonstrated that our proprietary DNA elements, Selexis Genetic Elements (SGEs), increase the transcription of a given transgene, thus boosting the overall expression of a therapeutic protein drug in mammalian cells . Howeverplus library displays a diversity of auxiliary proteins involved in secretory pathway machineries and cellular metabolism. Collectively, the libraries address a broad range of expression issues.Our specific approach involves CHO cell line engineering. We constructed CHO-M libraries based upon the CHO-M genome and transcriptome and using unique proprietary transposon vectors harboring SGE DNA elements to compensate for rate-limiting factors . Each CHplus libraries enabled the selection of a clonal cell line expressing 12 fold more product by comparison to the unmodified host cell [Figure ost cell .Our results demonstrate that components of the secretory and processing pathways can be limiting, and that engineering of the metabolic pathway ('omic' profiling) improves the secretion efficiency of therapeutic proteins from CHO cells.


Ovarian carcinomas consist of at least five distinct diseases: high-grade serous, low-grade serous, clear cell, endometrioid, and mucinous. Biomarker and molecular characterization may represent a more biologically relevant basis for grouping and treating this family of tumors, rather than site of origin. Molecular characteristics have become the new standard for clinical pathology, however development of tailored type-specific therapies is hampered by a failure of basic research to recognize that model systems used to study these diseases must also be stratified. Unrelated model systems do offer value for study of biochemical processes but specific cellular context needs to be applied to assess relevant therapeutic strategies.We have focused on the identification of clear cell carcinoma cell line models. A panel of 32 “ovarian cancer” cell lines has been classified into histotypes using a combination of mutation profiles, IHC mutation-surrogates, and a validated immunohistochemical model. All cell lines were identity verified using STR analysis.ARID1A and PIK3CA) and an overall molecular/immuno-profile typical of primary tumors. Mutations in TP53 were present in the majority of high-grade serous cell lines. Advanced genomic analysis of bona-fide clear cell carcinoma cell lines also support copy number changes in typical biomarkers such at MET and HNF1B and a lack of any recurrent expressed re-arrangements.Many described ovarian clear cell lines have characteristic mutations (including ARID1A and TP53 and a general immuno-profile serve well for establishing histotype of ovarian cancer cell We describe specific biomarkers and molecular features to re-classify generic “ovarian carcinoma” cell lines into type specific categories. Our data supports the use of prototype clear cell lines, such as TOV21G and JHOC-5, and questions the use of SKOV3 and A2780 as models of high-grade serous carcinoma.Conclusions: As with primary ovarian tumors, mutation status of cancer genes like  Ovarian cancer is a diverse set of diseases and amongst the most clinically significant, epithelial ovarian cancers (EOC), at least five distinct entities exist BRCA1/BRCA2 mutations also appear largely restricted to this histology Clinical responses and epidemiological differences are also apparent between histotypes. High-grade serous cancers show the best initial response rates to the current standard chemotherapy regime of platinum and taxanes Regardless of origin or histological similarities and differences, biomarker and genomic studies have been successfully used to distinguish each histotype and may represent a far more biologically relevant basis for classifying and subsequently treating EOCs. Although this concept is well-accepted, and gaining traction on becoming a new clinical standard, ambiguous cell line models perpetuated through molecular biology bench research hamper the development of tailored type-specific therapies. Those using bench experiment model systems must recognize that, like primary cancers, the models used to study these diseases must also be stratified. Although biochemical studies can generate useful information from using a variety of unrelated model systems, disease specific studies need to apply cellular context. The vast majority of research employing functional studies on “ovarian cancer” cell lines does not properly ascertain the background of their model systems. Resulting conclusions may be difficult to interpret and the value of potential therapeutic targets may be questionable as is the true relevance to a particular disease.Cell line studies of ovarian cancer have been severely hampered due to the lack of proper annotation of “ovarian” carcinoma cell lines. Once in culture, cells no longer have easily identifiable morphological traits to aid in histological classification. Additionally, human error, mislabeling and the generic feature of “epithelial-like” cell lines have also led to mix ups of cell lines and contamination which has resulted in un-interpretable data 2. See in vitro culture of primary patient material obtained through the OVCARE Tumor bank. All patients with tissue deposited in the OVCARE tumor bank provided written consent for experimental studies including sequencing, IHC characterization, and derivation of long-term cell lines from tissue samples. The OVCARE tumor bank study was approved under University of British Columbia and British Columbia Cancer Agency Research Ethics Board H05-60119 protocol.Cells were maintained in a humidified incubator at 37C with 5% COAll cell lines were subjected to identity testing using STR genotyping  at the College of American Pathologist's (CAP) accredited Centre For Translational and Applied Genomics (CTAG) as per manufacturer directives. Only lines with profiles matching public repository records, reported STR Cell lines were scraped from culture plates, washed 2× with PBS and pelleted. Cell pellets were re-suspended in ∼500 μl 10% Neutral Buffered Formalin (NBF) and allowed to fix overnight. Cells were pelleted again and re-suspended in a Histo-gel (Thermo-Fisher) plug prior to embedding in paraffin. A tissue microarray (TMA) was constructed as previously described ACTB, SDHA, RPL19, POLR1B, PGK1) as per manufacturers directives. In the case of TFF3 mRNA levels we considered any sample with detectable transcripts to be positive and substituted a score of “1” in place of TFF3 IHC when using COSP. The detection threshold (DT) for mRNA was considered to be the maximum count from spike-in negative control probes  plus 2 standard deviations. Statistical tests were calculated using GraphPad Prism v6.0c software.RNA was extracted from cell lines using Qiazol-miRNeasy kit (Qiagen) protocol and from primary tumors, 12 randomly selected from each histotype, using the miRNeasy FFPE kit (Qiagen). All RNA transcript levels were measured using the NanoString nCounter system ARID1A was done through a combination of custom hybrid capture and transcriptome sequencing on an Illumina GAII next generation sequencing (NGS) system as described previously Genomic DNA was extracted using standard methods . Regions encompassing mutations of known significance (Cancer hotspots) were Sanger sequenced using M13-tagged primers. Sequencing of DNA copy number was inferred from Affymetrix SNP 6.0 genome-wide microarrays. Arrays were run as per manufacturers directives and copy number ratio generated from an unpaired reference. Detection of copy number changed regions was done using a segmentation algorithm. All analysis and visualization was executed with Partek Genomics Suite 6.6, raw data is available from NCBI GEO [Accession GSE48351].TFF3 mRNA in primary samples appeared similar to that reported by IHC TFF3 mRNA for IHC and scored any cell line with detectable mRNA as “1” in our COSP algorithm . Given the strong negative association of mutation status and detectable protein expression BRAF, KRAS, NRAS, ERBB2, EGFR, CTNNB1, PIK3CA, PPP2R1A and DICER1. All coding exons of TP53 were verified in all cell lines. ARID1A mutations were tested using a custom NGS gene hybrid capture strategy ARID1A data from COSMIC and CCLE in addition to IHC as an ARID1A mutation-testing surrogate  and a mutation of unknown significance in TP53 (p.Y126C (het)), though p53 expression by IHC appeared normal. The 2008 cell line had undetectable ARID1A, suggesting loss of function, and also carried two TP53 mutations (c.572_574 delCTC (het)/c.673-1 G>T ) and corresponding p53 IHC overexpression. These atypical combinations of mutation could plausibly be explained by a propensity to accumulate mutations in cell lines with DNA mismatch repair (MMR) deficiencies, as has been reported for IGROV1, SKOV3, and A2780 We took note that a number of cell lines often used as high-grade serous models or generically as “ovarian carcinoma” had both As our primary objective was to describe CCC cell lines we generated copy number profiles of bona-fide CCC cell lines using Affymetrix SNP6.0 microarrays. Consistent with previous reports using primary tumor samples, CCC lines showed a moderate degree of copy number abnormalities, suggesting a genome that has undergone some degree of genomic instability.HNF1B (5/7) and MET (4/7), including one with high-level amplification (JHOC-5), similar to previous reports for CCC tumors ERBB2, in all cases the amplicon segment also encompassed the nearby CCC biomarker HNF1B, and none were positive for HER2 protein expression by IHC (not shown). Copy number loss around TP53 was observed only in a single CCC cell line  and, as noted above, all CCC lines appeared to have a normal-like expression pattern for p53 (IHC score 1).A limited number of literature reports have highlighted genes with mutations, overexpression and/or amplification amongst primary CCC, some with a relationship to survival or advanced disease As with other ovarian carcinoma types, recurrent translocations amongst CCC have not been described, though only a minimal number of studies have been undertaken ARID1A mutations were prevalent in these cell lines. Although three CCC lines did not have identifiable ARID1A mutations, only JHOC-5 cells appeared to have both wild-type sequence and detectable protein expression. The number of ARID1A “normal” CCC lines is lower than might be expected given the frequency of ARID1A mutations (and negative IHC) observed in primary CCC ARID1A null CCC lines to adapt to in vitro culture. However, given the small sample size it may well be a chance occurrence and does not appear to be significant. Other detected mutations  are all consistent with varying frequencies in CCC. TP53 mutations are notably absent in all of our validated CCC cell lines, as a de-facto defining characteristic, and only a single CCC line had heterozygous copy number loss though still retained normal-like p53 IHC.As our initial goal was identification of bonafide CCC cell lines, we are pleased to report that the majority of reported CCC lines were representative of the primary tumors' molecular and pathological phenotype. Our immuno-classification scheme, COSP, predicted most to be CCC and our own mutation data, as well as that from COSMIC and CCLE, suggested loss of function ARID1A and PIK3CA, are common to both types, overall supporting a related origin and similar route to transformation TP53 mutations, atypical for true CCC. IHC was negative for ARID1A, supporting a non-serous origin. We favored an assignment of ENOCa base largely on the TP53 mutation though note that this cell line is quite atypical as it may carry loss of function changes for ARID1A, mutation of TP53, and is positive for the CCC biomarker HNF1B. Arguably errors in cell line histotype reports may be explained simply by historically poor reproducibility in cell type assignment, though it is not unforeseeable that the biological relationship between CCC and ENOCa could be influencing these phenotypes. Given the high degree of overlap between the mutational characteristics of CCC and ENOCa, our panel was not able to further segregate or clarify this apparent confusion. SKOV3 is another unique case as it's immuno-phenotype most closely resembles HGSC, yet it caries a truncating mutation for ARID1A, a mutation that has not been observed in HGSC despite widespread testing PIK3CA mutation. Finally, the TOV112D cell line also presents as an exception with a moderately strong prediction of HGSC immuno-phenotype. In spite of this finding we suggest this line is representative of TP53 mutant ENOCa, based on the presence of an ENOCa characteristic CTNNB1 mutation, pathological review of the primary tumor material in the originating laboratory and expression profiling experiments supporting this conclusion Both CCC and ENOCa appear to arise in a background of endometriosis. Atypical endometriosis adjacent to, or contiguous with, either histotype is not unusual for either CCC or ENOCa NRAS mutant LGSC tumor and the first validated LGSC derived cell line carrying this mutation. The COSMIC database suggests the cell lines LK-1  and TYK-nu  also carry activating NRAS mutations, however we were unable to source these cell lines to confirm/reject their histological identity. In the cases of LGSC cell lines derived in-house, mutations of TP53 were not observed, consistent with IHC based literature reports suggesting this is a major molecular discriminator between HGSC and LGSC Unfortunately our COSP tool is unable to differentiate LGSC. Based on expert re-review of primary material we are aware of two cell lines derived from LGSC primary tumors. We therefore confidently favor this classification for VOA1056_CL and VOA1312_CL despite predictions of HGSC or ENOCa obtained from COSP. The VOA1056_CL line carries a Ras-pathway mutation as might be expected of an LGSC tumor, however this is an NRAS Q61R activating mutation. Activating NRAS mutations were recently described in LGSC at the 2012 AACR annual meeting TP53 homozygous deletion, overexpression by IHC, and KRAS G12V mutation.Finally only a single cell line in our collection was reported to be of mucinous carcinoma origin. The mutation profile of this cell line is consistent with this diagnosis, including a 127-bp Cell line records for epithelial ovarian carcinoma have recently come into question with a number contaminated and redundant cell lines acknowledged in a recent study In the spectrum of ovarian carcinomas, recent evidence strongly supports diagnosis and treatment of the five major histotypes of carcinomas as distinct diseases. Cancer cell lines provide an important intermediate tool for clinically relevant translational science, allowing genomic manipulation and cell biology studies beyond what can be reasonably achieved in clinical trials or animal models of cancer. In order to develop appropriate treatments, translational researchers need to use model systems appropriate to each ovarian carcinoma type. Unfortunately, historical records of ovarian cancer cell lines have rarely included information on histological origin Figure S1127bp homozygous (or hemizygous) deletion affectingTP53exon 4 in the MCAS mucinous carcinoma cell line. This mutation was apparent by Sanger sequencing though not annotated in the CCLE database. Coding bases are annotated in upper case.(PDF)Click here for additional data file.Table S1Cell Lines & Sources.(PDF)Click here for additional data file.Table S2Antibodies and Dilutions.(PDF)Click here for additional data file.Table S3deFuse predicted expressed re-arrangements from transcriptome sequencing data.(XLS)Click here for additional data file.Table S4Mutations Found In Ovarian Carcinoma Cell Lines.(PDF)Click here for additional data file.Table S5Mismatch Repair IHC.(PDF)Click here for additional data file.