We have investigated the affinity and kinetics of 125I-EGF – EGFR interaction is greatly dependent on cell line at normal culturing conditions, ranging from KD≈200 pM on SKBR3 cells to KD≈8 nM on A431 cells. The 125I-EGF – EGFR binding curves (irrespective of cell line) have strong signs of multiple simultaneous interactions. Furthermore, for the cell lines A431 and SKOV3, gefitinib treatment increases the 125I-EGF - EGFR affinity, in particular when the cells are starved. The 125I-EGF - EGFR interaction on cell line U343 is sensitive to starvation while as on SKBR3 it is insensitive to gefitinib and starvation.Highly repeatable and precise measurements show that the overall apparent affinity of the The intriguing pattern of the binding characteristics proves that the cellular context is important when deciphering how EGF interacts with EGFR. From a general perspective, care is advisable when generalizing ligand-receptor interaction results across multiple cell-lines. Cells are complex units with a heterogeneous surface. It is therefore likely that a ligand interacting with a cell binds to more than one receptor. These receptors may be members of the same receptor family or even the same receptor in different conformations. Nevertheless, the common discussion about biomolecular interactions tends to be simplistic. It is often assumed that most interactions are so called 1∶1 interactions, with one monovalent ligand binding to one specific target Signs of heterogeneity have been described for the epidermal growth factor (EGF) interacting with the EGF receptor (EGFR). Previous results indicate two receptor populations: one binding with high affinity (10–100 pM) and one with low affinity (1–10 nM) Atypical expression or activity of EGFR is present in numerous kinds of cancer 125I-EGF-EGFR interaction in four cancer cell lines. We searched for signs of multiple interactions occurring simultaneously and compared the affinity. The cells were exposed to four different treatments and gefitinib sensitivity and impact of starvation of the cell lines were studied.The aim of this study was to provide new information on the intricate interaction pattern of EGF and EGFR by comparing the kinetics and the affinity of the a (M−1s−1) is the association rate constant and kd (s−1) is the dissociation rate constant describing the formation and dissociation of the complex. The amount of complex can be rewritten as tot is the total amount of receptors (bound and unbound) and KD is the equilibrium dissociation constant (often denoted affinity). KD is in turn the ratio between kd and ka,Since the reversible 1∶1 interaction model is the general choice when discussing biological interactions, we will start with the same assumption. It can be described byD, 1×KD or 10×KD, the amount of bound receptors at equilibrium will be 10%, 50% or 91% respectively.Equation (3) can be utilized when calculating the amount of receptors bound at a specific concentration of the ligand L. For example, if using a ligand concentration of 0.1×KAt steady-state, f(t) = 1. In a biological context, the effect of an interaction often occur long before equilibrium has been reached (because time to equilibrium can be many hours or days) and the time for the effect to remain is often due to the dissociation time d, the slower the dissociation”, which combined with (4) means that a slow dissociation also is a sign of a low KD, i.e. a high affinity. Equation (5) is more difficult to grasp, as both ka and kd are involved, but in general a higher affinity will result in less curvature and longer time to equilibrium.Equations (5) and (6) describe how the affinity and the curvature of a real-time binding trace are related. Equation (6) can be translated as “the lower the kD of the 125I-EGF – EGFR interaction. The signal levels from the end of each 125I-EGF incubation, representing approximately steady-state, were analyzed using a non-linear regression model describing a 1∶1 interaction. Calculations of KD were made using data from two titration measurements for each cell line. A 1∶1 interaction model is not sufficient to describe the system, as 125I-EGF seems to bind to the cells in a multiple manner (see below), but the model was still chosen to provide results comparable with common saturation curve analysis in literature.Data from stepwise titration affinity experiments was used to estimate the equilibrium dissociation constant K125I-EGF – SKBR3 interaction had the highest affinity, with a KD value of approximately 0.2 nM. 125I-EGF binds to SKOV3 and U343 cells with an intermediate strength, with KD values of approximately 0.9 and 1.4 nM respectively. A431 cells show the weakest interaction, with a KD of as much as approximately 8 nM. The difference of a factor 40 between the strongest and the weakest interaction came as a surprise, as the 125I-EGF – EGFR interaction was expected to be the same no matter the cell. To our best knowledge, there are no known mutations on the extracellular part of EGFR in any of the four cell lines that could affect the affinity.125I-EGF – EGFR interaction the data showed signs of multiple interactions taking place, observed as binding curvatures different from ordinary 1∶1 interactions. This was concluded by fitting the binding curves from the affinity titration to models describing one monovalent ligand (125I-EGF) binding to either one or two independent receptors on the cell with different kinetic parameters. Representative data from one A431 and one U343 affinity measurement is depicted in When evaluating the kinetics of the 6 receptors/cell and 5×105 receptors/cell respectively) One possible explanation for the large differences in affinity and the multiple simultaneous interactions is the presence of EGFR homo- and hetero-dimers. Varying EGFR and HER2 receptor expression levels could lead to different proportions in the homo/hetero-dimer population and this could play a significant role for the interaction pattern and apparent affinity for a particular cell line. A431 and U343 over-express EGFR  prior to and during the measurements. Uptake measurement consisted of a two-step incubation, with pairs of concentrations chosen to receive a clear increase in signal by the second addition of 125I-EGF. As the affinity study proved that the nature of the 125I-EGF – EGFR interaction varies greatly between the cell lines, the concentration pairs for the measurement had to be chosen differently for the cell lines to obtain comparable results for the treatment effect analysis.The uptake and retention of Normalized interaction data for each treatment of A431 shows good reproducibility for the method . To enha125I-EGF – A431 interaction is clearly affected by the presence of gefitinib, in particular in serum free medium. Gefitinib treated cells have a smaller difference in signal level between the first and second concentration  is close to KD which makes the detected output highly sensitive to any affinity changes. The measurements in this paper were conducted at room temperature, and no signs of internalization were detected for the U343 cell line during a 24 hour timeframe (data not shown) implying that internalization can be neglected in all our experiments. Thus, gefitinib binding to the intracellular part of the EGF receptor can apparently change the extracellular binding properties of EGFR. If this effect is due to conformational changes of the receptor or intracellular processes is yet to be determined, although the increased effect of gefitinib in starved A431 and SKOV3 cells indicate that the condition of the cell is also important.All in all, gefitinib can affect the binding of The surprisingly large difference in affinity of EGF-EGFR across different cell lines and treatments gives reason for reflection. Even though we have only investigated one ligand-receptor interaction in detail, we cannot exclude that the hosting cell line influences the interaction results also for other receptors. In our laboratory, we will continue to investigate various ligand-receptor interactions across different cell lines to better understand the importance of cellular context.By use of data from repeatable, high-precision interaction measurements, we conclude that at normal conditions, EGF interacts with EGFR in quite different manners depending on the hosting cell line. It is further evident that the addition of gefitinib or the lack of serum in the cell-culture medium can alter the EGF-EGFR interaction properties at least 10-fold, but again depending on the hosting cell line.We have clear indications on that the apparent EGF-EGFR interaction is composed of multiple different interactions and that the affinity varies greatly between the cell lines, but more work is required to decipher the underlying nature of the EGF-EGFR interaction. Thus, the EGF receptor family, its multiple interactions with EGF and the effects of intracellular effects remains an unsolved puzzle with need for further investigation.From a general perspective, it may be advisable to limit the discussion of any ligand-receptor interaction to the context of the hosting cell-line and care is recommended when generalizing interaction results across multiple cell-lines.2.The human squamous carcinoma cell line A431 , the human glioma cell line U343MGaCl2:6 (denoted U343), the human ovarian carcinoma cell line SKOV3  and the human breast cancer cell line SKBR3  were used in the experiments. The cells were seeded on a local area of a cell culture dish , as described previously 125I  using the Chloramine-T protocol 2.5 µg human Epidermal growth factor  was labeled at four occasions with 10–20 MBq 125I-EGF to seeded cells were monitored in real-time at room temperature using LigandTracer Grey, as described preciously by Björke and Andersson 125I-EGF in complete medium was added in each affinity titration assay. The measurements were performed in duplicates for each cell line and different concentration series were used to fully cover the concentration span needed for a proper affinity estimation  and fitted to kinetic interaction models describing a monovalent ligand binding to either one (1∶1) target or two (1∶2) independent targets.D.The estimated steady-state signals from both affinity measurements were analyzed simultaneously in MATLAB 6.5  and a 1∶1 steady state interaction model was fitted using non-linear regression to obtain the equilibrium dissociation constant, KIn order to enhance visibility of the acquired binding curves, a moving window Fourier transform noise reduction algorithm was applied. Noise with a frequency higher than approximately 0.01 Hz was removed, resulting in noise-reduced curves where the biologically relevant curvature was preserved.Four kinds of treatment of the cells were tested: (a) in normal cell culture medium, as described above, (b) in cell culture medium devoid of fetal calf serum, (c) in cell culture medium supplemented with 1 µM gefitinib  and (d) in cell culture medium devoid of serum but with 1 µM gefitinib. The cell culture medium of the starvation dishes (b) were exchanged with serum free Ham's F10 24 h prior to measurement. The gefitinib dishes (c) were incubated with 1 µM gefitinib 48 h before measurement. Fresh medium with 1 µM gefitinib was added after 24 h to ensure a continuous gefitinib exposure. Prior to retention measurements, more gefitinib was added to ensure a fresh gefitinib concentration of at least 1 µM. The gefitinib +starvation dishes (d) were treated similar to (c), but with serum free medium +1 µM gefitinib added 24 h prior to measurement.125I-EGF (Detections of treatment effects were performed at room temperature using LigandTracer Grey. All measurements were conducted in 3 ml cell culture medium and started with a short baseline, followed by a two-step uptake study using increasing concentrations of 125I-EGF , followe


Cell line models have proven to be effective tools to investigate a variety of ovarian cancer features. Due to the limited number of cell lines, particularly of the serous subtype, the heterogeneity of the disease, and the lack of cell lines that model disease progression, there is a need to further develop cell line resources available for research. This study describes nine cell lines derived from three ovarian cancer cases that were established at initial diagnosis and at subsequent relapse after chemotherapy.in vivo tumorigenicity potential of the cell lines was measured. Response to paclitaxel and carboplatin was assessed using a clonogenic assay.The cell lines from three women diagnosed with high-grade serous ovarian cancer  were derived from solid tumor (TOV) and ascites (OV), at specific time points at diagnosis and relapse (R). Primary treatment was a combination of paclitaxel/carboplatin , or cisplatin/topotecan (2295). Second line treatment included doxorubicin, gemcitabine and topotecan. In addition to molecular characterization , the cell lines were characterized based on cell growth characteristics including spheroid growth, migration potential, and anchorage independence. The 50 values and dose response curves, there was clear evidence of acquired resistance to carboplatin for TOV2295(R) and OV2295(R2) cell lines.All cell lines had either a nonsense or missense TP53 mutations. The ability to form compact spheroids or aggregates was observed in six of nine cell lines. Limited ability for migration and anchorage independence was observed. The OV3133(R) cell line, formed tumors at subcutaneous sites in SCID mice. Based on ICThe study identified nine new high-grade serous ovarian cancer cell lines, derived before and after chemotherapy that provides a unique resource for investigating the evolution of this common histopathological subtype of ovarian cancer. Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy. The most common histopathogical subtype, serous, accounts for at least 50% of EOC ,2. OvariCell line models have proven to be effective tools in ovarian cancer research and have been utilized to investigate the molecular and cellular features of ovarian cancer ,12. We hin vivo capacity to form tumors in SCID mice. Mutational status for genes important in serous EOC such as TP53, BRCA1 and BRCA2 were investigated. Information on disease progression and treatment regimens are included. In addition, we have characterized the chemosensitivity of the cell lines to paclitaxel and carboplatin by clonogenic assays. These cell lines provide novel and comprehensive models for the study of EOC progression, ascites formation and resistance to chemotherapy.This study evaluated ovarian cancer cell lines derived from the same patient at diagnosis and at relapse following exposure to chemotherapy. The cell lines were developed from solid tumors and ascites. Nine cell lines were developed from specimens obtained from three patients diagnosed with high-grade serous ovarian cancer. The cell lines were characterized biologically by growth rates, morphology, ability of forming three-dimensional spheroids, migration and invasion potential, and their Tumor and ascites samples were collected with informed consent from the Centre hospitalier de l’Université de Montréal (CHUM), Hôpital Notre-Dame, in the Department of Gynecologic Oncology. The study was approved by the Comité dé’thique de la recherché du CHUM, the institutional ethics committee. Stage was determined at time of surgery by a gynecologic oncologist. Histopathology and tumor grade were determined by pathology using criteria consistent with the International Federation of Gynecology and Obstetrics (FIGO) classification ).2, and 5% CO2 and grown in complete OSE medium, which includes OSE medium , 10% FBS, 0.5 μg/mL amphotericin B  and 50 μg/mL gentamicin . The solid ovarian tumor (TOV) derived cell lines , TOV3133G and TOV3133D) were established using the scrape method as previously described [In total, nine cell lines were derived from three patients. The patients were coded with the unique identifiers 1369, 2295 and 3133. All cell lines were maintained in hypoxic condition of 5% Oescribed ,24,25. B5 cells per well for the 1369 and 2295 cell lines and 2 x 105 cells per well for 3133 cell lines). The OSE complete media was replaced every 3 days for the duration of the experiment. Every second day from day 1 to 13, cells were trypsinized, resuspended in media and counted using the CASY analyzer system. Saturation density was defined as the mean maximum number of cells counted at the time of confluence. Each experiment was performed in duplicate, and repeated three times. Doubling times were determined using a publically available algorithm [Growth rates were assessed as previously described ,19. Brielgorithm .Western Blot (WB) and immunohistochemistry (IHC) analyses were performed using the following antibodies: beta actin ; TP53 (D0-1) ; HER2/ErbB2/Neu (C-18) ; Keratin 7 Ab-2 ; Keratin 8 Ab-4 ; Keratin 18 (DC-10) ; Keratin 19 Ab-1 , and Keratin 20 (SPM140) .2O2 treatment was used to eliminate endogenous peroxidase activity. The sections were blocked with a protein blocking serum-free reagent  and incubated with the antibody used for western blotting for 60 min at room temperature. Tissues were incubated with a secondary biotinylated antibody (DakoCytomation) for 20 min followed by incubation with a streptavidin-peroxidase complex (DakoCytomation) for 20 min at room temperature. Staining was visualized using diaminobenzidine containing a peroxidase substrate (DakoCytomation). Hematoxylin was used as the counterstain and all sections were observed by light microscopy and pictures were taken at 40x magnification. Substitution of the primary antibody with phosphate buffered saline served as a negative control.Tissue sections were fixed in formalin and embedded in paraffin blocks. Sections (4 μm thick) were cut and slides were stained using the immunoperoxidase method. Tissue sections were heated (37°C overnight or 60°C for 30 minutes), deparaffinized in toluene and rehydrated in ascending concentrations of ethanol. Slides were then heated in boiling citrate buffer  to unmask antigens. A 0.3% HDNA was extracted from cell lines as described previously . TP53 mu3 cells were suspended in 16 μl of complete OSE medium and placed on the cover of non-coated plastic tissue culture plates that were subsequently inverted. Phosphate buffered saline (1 x PBS) was added to the bottom plate to prevent dehydration of droplets. Spheroid formation ability was assessed in complete OSE medium after four days of incubation at 37°C, 5% O2, 5% CO2, with spheroid formation of the cell lines being classified concordant with previous research [A spheroid assay was conducted to determine the ability of cell lines to generate three-dimensional structures in the form of aggregates, as previously described ,21. Brieresearch ,21.4 cells in a semi-solid media containing noble agar. Cells were included in the top layer formed of 0.33% w/v agar in complete OSE medium that was applied over a base layer (0.66% w/v agar in complete OSE medium) [Cell lines were assayed for their ability to grow in anchorage independent conditions by culturing 2 x 10 medium) . Cells wMigration potential was evaluated using the scratch assay method as previously described ,19,29. C6 cells resuspended in 100 μl of cold phosphate buffered saline (D-PBS)  and 100 μl of Matrigel . The animals were housed under sterile conditions in a laminar flow environment with ad libitum access to food and water. Tumor formation was assessed twice a week for over 100 days. Animals were sacrificed before neoplastic masses reached limit points established by the Institutional Committee on Animal Protection (CIPA) according to the Canadian Council on Animal Care.The tumorigenic potential of cell lines was assessed based on their ability to form tumors in 50 day-old female SCID (severe combined immunodeficiency) mice, and NOD SCID  at subcutaneous left gluteal injection sites. A volume of 200 μl was injected in each mouse and consisted of 5 × 103 cells/well; OV2295(R2), 2 × 103 cells/well; TOV2295(R), 2 × 103 cells/well; TOV3133G, 2.5 x 103 cells/well; TOV3133D, 1 × 103 cells/well; OV3133(R), 1 × 104 cells/well; OV3133(R2), 5 × 104 cells/well). Cells were seeded and allowed to adhere for 16 hours in a 37°C, 5% CO2, 5% O2 incubator after which the media was removed and replaced with OSE complete media containing paclitaxel (0–300 nM), or carboplatin (0–300 μM) . Cells were incubated with the drug for 24 hours. The drug was then removed, cells were washed with 1 x PBS and OSE complete media was added. Media was changed weekly until colonies were visible. Colonies were then fixed with cold methanol and colored with Giemsa . Colonies were manually counted and reported as percent of control. IC50 values were determined using Graph Pad Prism 3 software . Each individual experiment was performed in triplicate and repeated three times.Chemotherapy sensitivity of cell lines was assessed using a clonogenic assay . BrieflyPatient 1369 was previously diagnosed with breast cancer 18 months prior to her ovarian cancer diagnosis. Following surgical resection for breast cancer, she received docetaxel treatment, which was completed 14 months prior to her ovarian cancer diagnosis. She also entered a clinical trial, NSABP B-30 BRAS1 . She also received radiotherapy 12 months prior to her ovarian cancer diagnosis. After ovarian cancer diagnosis, she received a treatment of paclitaxel and carboplatin for 5 months. Based on the serum CA-125 levels  for the first four months following diagnosis during which period her CA-125 levels decreased significantly, from 6000 units/ml to lower than 50 units/ml during days 96 through 200  cystadenocarcinomas, of the serous papillary histopathology  were present in the protein extract of each of the nine cell lines Figure A. ExpresEach of the cell lines harbored a TP53 mutation as verified by sequence analysis Table . The mutThe growth rates of the cell lines was determined Table  and compThe cell line’s ability to form spheroids was measured using the hanging droplet method as previously described . AlthougThe migration potential of the cell lines was measured using an established scratch migration assay. There were no notable differences in migration rates between the cell lines. All cell lines migrated slowly and did not fill the gap within 48 hours, which is slower than what was observed with TOV1946 [Using the soft agarose assay, the anchorage dependency of the cells lines was investigated. After three weeks there were visible colonies formed with the OV1369R2), OV2295(R2), TOV2295(R), TOV3133D and OV3133(R2) cell lines  (5), TOV1946 (3) and TOV112D (3)). Only OV3133(R) formed tumors in SCID mice (66.7% of mice), whereas all other cell lines failed to induce any tumor formation (Table 3 for OV3133(R) after 85 days whereas those with TOV112D and TOV1946 reached 3000 mm3 in less than 30 days. All other cell lines formed masses that remained at 100 mm3 or less for the length of the experiment , TOV50 was calculated from these curves to allow comparison between the cell lines. Data from previously published cell lines, TOV112D and TOV1946, are included in Figure The cell lines were investigated for their sensitivity to carboplatin and paclitaxel by determining a dose response curve Figure  obtained50 values were much higher than other cell lines studied for carboplatin  but more comparable to the values obtained with TOV112D and TOV1946 . For paclitaxel, the IC50 values of TOV1369 (22.8 ± 10.7 nM) and OV1369(R2) (9.13 ± 6.88 nM) suggest a lower sensitivity to the drug when compared to the other cell lines tested ), IC), IC50 v46 Table . In the 50 calculated for carboplatin for the two post-chemotherapy cell lines, OV2295(R2) and TOV2295(R)  were more than ten fold higher than the IC50 of the pre-chemotherapy cell line OV2295 (0.05 ± 0.01 μM). In the case of paclitaxel, values were similar for the three 2295 cell lines (p > 0.05) where the determined IC50 for OV2295, OV2295(R2) and TOV2295(R) were 5.43 ± 4.73, 1.99 ± 1.48 and 1.87 ± 0.31 nM, respectively. These results suggest that patient 2295 acquired resistance to carboplatin, but not to paclitaxel.For 2295, the IC50 for the 3133 cell lines ranged between 0.75 ± 0.63 μM (TOV3133G) to 2.65 μM (OV3133(R2)) for carboplatin, and from 1.59 nM (OV3133(R2)) to 5.54 ± 3.19 nM (OV3133(R)) for paclitaxel. Note that the IC50 value for OV3133(R2) was based on one experiment conducted in triplicate due to the low clonogenic efficiency of OV3133(R2). There was no difference in IC50 values between the pre- and post-chemotherapy cell lines derived from patient 3133 for either carboplatin or paclitaxel.For the pre- and post-chemotherapy cell lines derived from patient 3133, there was no notable difference in chemosensitivity for either carboplatin or paclitaxel Figure , Table 2We report here on matched EOC serous cell lines derived from solid tumor or ascites samples from the same patient at time of diagnosis and following recurrence. Ovarian epithelial cells typically express keratin 7 but lack expression of keratin 20 -33. ThisThe cell lines were characterized in terms of TP53 mutation status and protein expression, BRCA1, BRCA2, KRAS and BRAF mutation status, and HER2 expression. All of the cell lines had a somatic TP53 mutation, which is consistent with the reported frequency in high-grade serous tumors, estimated at 51 to 93% in recent studies -37. An iin vivo tumor setting [The distinct tumor growth characteristics within the serous cell lines derived here, indicates a diversity reflective of the heterogeneous nature of this histopathological subtype. For example, saturation density, spheroid formation and colony formation in soft agarose differed between cell lines, and also within cell lines derived from the same patient. Differences in spheroid formation between cell lines derived before and after chemotherapy treatment may offer an interesting point of reference, especially as spheroid models may offer a model system more in line with the  setting . For exa setting  and the  setting . In addi setting .in vivo tumor formation of the cell lines in SCID mice at subcutaneous sites was only observed with OV3133(R), the first ascites taken from patient 3133. The second ascites sample OV3133(R2) that was taken after doxorubicin treatment, at approximately 500 days after the OV3133(R) was sampled, did not form tumors. The tumors formed in SCID mice grew slowly as compared with the previously established TOV112D and TOV2295(R) cell lines, although this observation was consistent with previously studied high grade serous cell lines such as TOV2223 which also did not form tumors at subcutaneous sites in SCID mice [Interestingly, CID mice . Althougin vitro, which are often derived from clonal variants that survive by escalating dosages of chemotherapeutics. In contrast, the cell lines we describe here may more accurately represent the molecular evolution that occurs within the tumor microenvironment. Additionally, we have already alluded to the potential to further study the cell lines by utilizing the spheroid model, which may more accurately reflect the in vivo tumor environment [The new cell lines derived in this study were developed from patient samples that were exposed to specific chemotherapeutic agents. This is in contrast to chemoresistant cell lines generated ironment . For exaironment . Interesironment . This su50 levels of paclitaxel response in TOV1369 and OV1369(R2) are four-to-twenty and two-to-five times higher, respectively, than all other cell lines examined, and this is possibly due to acquired resistance to taxol as a consequence of prior treatment for breast cancer. We also note that cells from patient 1369 also displayed a lower sensitivity to carboplatin, although the clinical profile of this patient does not suggest inherent chemoresistance. Comparison of the OV2295 (derived prior to recurrence) to the OV2295(R2) and TOV2295(R) cell lines derived following recurrence were the only clear example of acquired resistance to carboplatin. Carboplatin resistance is well documented in ovarian cancer. For example, a recent study found 75% of solid tumors and 59% of ascites samples to be resistant to carboplatin [Of the patients from which the cell lines were established, two of the three (1369 and 2295) responded initially to first line therapy using Response Evaluation Criteria in Solid Tumors (RECIST) criteria ,47. In gboplatin . This isMutation status such as TP53, BRCA1 and BRCA2 ,51 are aThe new ovarian cancer cell lines characterized in this report provide an important biological resource for studying the molecular genetic evolution of ovarian cancer that reflect the development of disease in the context of initial diagnosis and following disease recurrence. The cell lines provide a framework for comparative molecular genetic studies investigating the genomic landscape by gene expression, copy number variation or mutation analysis. The unique phenotypes exhibited by the cell lines suggest that they reflect the complexity of ovarian cancer disease. The paired sample cell line model from patient 2295, will allow for further discrimination of acquired resistance, affected by the chemotherapy regime. Although cell lines from patient 1369 and 3133 do not show signs of acquired resistance, both offer avenues of research into tumor evolution. Specifically, further study of the1369 cell lines would allow for the understanding of the molecular basis for the high innate resistance to both carboplatin and paclitaxel. In 3133, three distinct time points are represented offering possible insights into the mechanisms of tumor progression and evolution.The authors declare that there are no competing interests.I.J.L. performed the clonogenic assays to assess chemosensitivity of the cell lines, contributed to the anchorage independent growth assay, participated in the xenograft tumor formation experiment and performed all data analysis and writing of the manuscript. M.C.J.Q. participated in all analysis, wrote the majority of the paper and was responsible for all editing of the manuscript. L.P. established the cell lines from tumor and ascites tissue. M.dL. and D.M.P. were responsible for all clinical data. L.W. performed the growth characteristic assays , data analysis of the migration assay and immunohistochemistry as well as participated in the xenograft formation experiment. K.Y.C. performed the Western blot and aided with the anchorage independent growth assay. L.C. and N.D. contributed to the clonogenic assay. L.M. helped perform the anchorage independent growth assay. Z.S. and S.L.A. performed the mutation analyses under the supervision of PNT. P.N.T., D.M.P. and A-M.M-M. were involved in the study conception and design, and interpretation of the data. All authors revised the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/379/prepub Keratin 7 and keratin 20 immunohistochemistry in ovarian tumors from which the cell lines were derived. Paraffin embedded colon tumors were used as the negative control for keratin 7, and positive control for keratin 20. Sections were counterstained with hematoxylin and are shown at x40 magnification. Click here for file Images from the migration assay, indicating migration of cells during a 48-hours period. Note the closure of the wound for the TOV1946 cell line after 48 hours.Click here for file Tumor growth in SCID mice following injection of specific ovarian cancer cell lines. For each cell line, six SCID mice received subcutaneous injection (5 x 106 cells mixed with Matrigel) (with the exception of five mice for OV1369(R2), and three mice for TOV112D and TOV1946). Graphs represent average ± SD of tumor volume as a function of days following cell injection. Tumor masses were measured at least twice a week (width x length x thickness). Note that the axis for TOV112D and TOV1946 is not of the same scale. Cell lines derived pre-chemotherapy are represented by a closed symbol, cell lines derived post-chemotherapy have an open symbol. Click here for file Tumor growth in SCID-NOD mice following injection of specific ovarian cancer cell lines. For each cell line, two NOD-SCID mice received subcutaneous injection (5 x 106 cells mixed with Matrigel). Graphs represent average of tumor volume as a function of days following cell injection. Tumor masses were measured at least twice a week (width x length x thickness). Note that the axis for TOV112D and TOV1946 is not of the same scale. Cell lines derived pre-chemotherapy are represented by a closed symbol, cell lines derived post-chemotherapy have an open symbol. Click here for file


Dermacentor reticulatus cell lines, embryo-derived primary cell cultures were set up from eggs laid by field ticks originally collected as unfed adults in The Netherlands and maintained for up to 16 months. After several months, it became evident that cells in the primary cultures were infected with a Rickettsia-like intracellular organism. Supernatant medium containing some D. reticulatus cells was inoculated into cultures of 2 Rhipicephalus (Boophilus) microplus cell lines, BME/CTVM2 and BME/CTVM23, where abundant growth of the bacteria occurred intracellularly on transfer to both cell lines. Bacterial growth was monitored by light  and transmission electron microscopy revealing heavy infection with typical intracytoplasmic Rickettsia-like bacteria, not present in uninfected cultures. DNA was extracted from bacteria-infected and uninfected control cultures, and primers specific for Rickettsia 16S rRNA, ompB, and sca4 genes were used to generate PCR products that were subsequently sequenced. D. reticulatus primary cultures and both infected tick cell lines were positive for all 3 Rickettsia genes. Sequencing of PCR products revealed 99–100% identity with published Rickettsia raoultii sequences. The R. raoultii also grew abundantly in the D. nitens cell line ANE58, poorly in the D. albipictus cell line DALBE3, and not at all in the D. andersoni cell line DAE15. In conclusion, primary tick cell cultures and cell lines are useful systems for isolation and propagation of fastidious tick-borne microorganisms. In vitro isolation of R. raoultii from Dutch D. reticulatus confirms previous PCR-based detection in field ticks, and presence of the bacteria in the tick eggs used to initiate the primary cultures confirms that transovarial transmission of this Rickettsia occurs.Tick cell lines play an important role in research on ticks and tick-borne pathogenic and symbiotic microorganisms. In an attempt to derive continuous  Hyalomma asiaticum cell cultures were more sensitive than chick embryo cell cultures for detection of very small amounts of tick-borne encephalitis virus. Similarly, Dermacentor variabilis cell lines to successfully isolate spiroplasmas from field-collected Ixodes pacificus ticks while axenic culture in specific spiroplasma broth failed. Inoculation of infected mammalian blood cells into I. scapularis cell lines has resulted in isolation and continuous culture of many obligate intracellular arthropod-borne bacterial pathogens, as reviewed by Tick cell lines play an increasingly important role in research on ticks and tick-borne pathogenic and symbiotic microorganisms . PrimaryDermacentor harbour several pathogenic and/or endosymbiotic Rickettsia species, including the human pathogens Rickettsia rickettsii, causative agent of Rocky Mountain spotted fever in North America, R. slovaca and R. sibirica in the Old World  and equine  babesiosis in many European countries. D. reticulatus ticks also transmit spotted fever group (SFG) Rickettsia species to humans  of the Faculty of Veterinary Medicine, Utrecht University (DEC No. 2008.II.07.068). Engorged females were sent to the University of Edinburgh where they were surface-sterilised  microplus embryo-derived cell lines BME/CTVM2 . The D. nitens ANE58 .The ME/CTVM2  and BME/ME/CTVM2  by standME/CTVM2 , were mans ANE58 , D. albis DALBE3 , and D. ni DAE15  embryo-dni DAE15  supplemeRickettsia-infected and uninfected control cell lines were fixed in 3% glutaraldehyde in cacodylate buffer for 2–3 h, post-fixed in 1% osmium tetroxide in cacodylate buffer, dehydrated in acetone, and embedded in Araldite resin. Sections were cut on a Reichert OMU4 ultramicrotome (Leica), stained in uranyl acetate and lead citrate, and viewed in a Phillips CM120 transmission electron microscope. Images were taken on a Gatan Orius CCD camera.In primary cultures and cell lines, cells were resuspended by gentle pipetting and approximately 50-μl aliquots used to prepare cytocentrifuge smears. The smears were air-dried, fixed in methanol, stained with Giemsa and examined using a Zeiss Axioskop 2 Plus microscope. Photomicrographs were taken using an AxioCam MRc digital camera and software. For electron microscopy, samples of resuspended cells from Total genomic DNA was prepared from infected and uninfected tick cells using the DNeasy Blood and Tissue Kit  following the manufacturer's protocol. Purified DNA was eluted from the spin column with 400 μl TE buffer (2 × 200 μl) and stored at −20 °C.ompB). A 900-bp fragment of the cell surface antigen sca4 gene was amplified using primers D1f (5′-ATG AGT AAA GAC GGT AAC CT-3′) and D928r (5′-AAG CTA TTG CGT CAT CTC CG-3′)  and Rick-R2 (5′-CAT CAC TCA CTC GGT ATT GCT GGA-3′) . The priC CG-3′) . Each 50www.dnaseq.co.uk using Applied Biosystems Big-Dye Ver 3.1 chemistry on an Applied Biosystems model 3730 automated capillary DNA sequencer. Homology searches of the 16S rRNA, ompB, and sca4 sequences in the NCBI database were performed using the BLAST search programme  following the manufacturer's recommendations. DNA sequencing in the forward and reverse directions was performed by DNA Sequencing & Services, MRCPPU, College of Life Sciences, University of Dundee, Scotland, rogramme . Sequencrogramme  for multompB, and sca4 genes of the Rickettsia isolated from one of the D. reticulatus primary cultures (DRET2) have been deposited in the GenBank database and assigned accession numbers JN242190, JN242189, and JN242188, respectively.The nucleotide sequences of the 16S rRNA, D. reticulatus, only 8 laid egg batches in which some or most of the eggs were fertile. These were used in 4 pools of 2 egg batches each to set up 11 primary cell cultures (1–4 cultures per pool). Five of the primary cultures were discarded within 10 weeks due to cell death or fungal contamination. The remaining 6 cultures (Rickettsia-like bacterium infecting varying proportions (<5 to >90%) of the cells in each culture  and BME/CTVM23 (n = 4) incubated at 28 °C, ANE58 (n = 3) and DAE15 (n = 3) incubated at 32 °C, and DALBE3 (n = 4) incubated at both temperatures. The bacteria grew vigorously in all BME/CTVM2 and BME/CTVM23 cultures .At 14 months, DNA was extracted from samples of cell suspension from the 6 primary cultures and selected subinoculated cell lines, and PCR-screened using 3  strains . All uniRickettsia genotypes were first identified by PCR and sequence analysis in D. nuttalli and Rhipicephalus pumilio ticks collected in Russia  microplus lines BME/CTVM2 and BME/CTVM23, and for the D. nitens line ANE58. A similar pattern was seen for R. peacockii  microplus cell line BME26, but grew poorly in DAE15 and DALBE3 cells. It is not known whether this difference in susceptibility to infection with Rickettsia spp. between individual cell lines is due to the species of origin or to the heterogeneous nature of uncloned, embryo-derived tick cell lines  cells  cells ; the bacechnique . Our resFrancisella, Midichloria, Coxiella, Diplorickettsia, and Arsenophonus (Francisella-like endosymbionts reported from ticks of the genera Dermacentor, Amblyomma, and Ornithodoros (D. andersoni cell line DAE100 was found to harbour R. peacockii (R. hoogstraali was detected in cell lines derived from the soft tick Carios capensis after 6 months in vitro (Rickettsia replication (R. raoultii in tick cell lines will aid in its characterisation, providing an excellent model system for study of the interaction between these bacteria and their arthropod vectors.Ticks have also been found to harbour a range of other endosymbiotic bacteria of genera including nophonus . Many ofthodoros . Moreoveeacockii , and R. in vitro . Tick celication . They arlication . The ava


Current protocols for the treatment of ovarian cancer include combination chemotherapy with a platinating agent and a taxane. However, many patients experience relapse of their cancer and the development of drug resistance is not uncommon, making successful second line therapy difficult to achieve. The objective of this study was to develop and characterize a cell line resistant to both carboplatin and docetaxel  and to compare this cell line to cells resistant to either carboplatin or docetaxel.The A2780 epithelial endometrioid ovarian cancer cell line was used to select for isogenic carboplatin, docetaxel and dual drug resistant cell lines. A selection method of gradually increasing drug doses was implemented to avoid clonal selection. Resistance was confirmed using a clonogenic assay. Changes in gene expression associated with the development of drug resistance were determined by microarray analysis. Changes in the expression of selected genes were validated by Quantitative Real-Time Polymerase Chain Reaction (QPCR) and immunoblotting.Three isogenic cell lines were developed and resistance to each drug or the combination of drugs was confirmed. Development of resistance was accompanied by a reduced growth rate. The microarray and QPCR analyses showed that unique changes in gene expression occurred in the dual drug resistant cell line and that genes known to be involved in resistance could be identified in all cell lines.Ovarian tumor cells can acquire resistance to both carboplatin and docetaxel when selected in the presence of both agents. Distinct changes in gene expression occur in the dual resistant cell line indicating that dual resistance is not a simple combination of the changes observed in cell lines exhibiting single agent resistance. Ovarian cancer remains the most lethal gynecological cancer, with a 5-year mortality rate greater than 50% . The higMechanisms underlying the development of resistance to platinating agents, especially cisplatin, have been well characterized and include repair of DNA lesions, translesional DNA synthesis, altered cellular transport of the drug, increased antioxidant production, and reduction of apoptosis -12. AlteThe difference in mode of action and mechanisms of resistance between platinating agents and taxanes is taken advantage of in dual agent chemotherapy of advanced ovarian cancer, to achieve significantly increased efficacy and progression free survival (PFS) of patients. The most common combination therapy is carboplatin together with paclitaxel, although the taxane docetaxel has also been used with similar efficacy -28. NotwTo investigate if the development of dual agent resistance invokes different mechanisms or is a combination of the mechanisms of resistance that arise upon exposure to single agents, we have developed a set of isogenic ovarian cancer cell lines resistant to either carboplatin, docetaxel or a combination of carboplatin and docetaxel. Changes in gene expression associated with the specified drug resistance in each cell line were analyzed using microarray analysis. Comparison between the three resistant cell lines permitted identification of shared and different changes in gene expression among the cell lines. This analysis showed that the establishment of carboplatin and docetaxel resistance does not share many changes in gene expression and that dual agent resistance appears to develop from mostly unique changes in gene expression, different from both carboplatin and docetaxel resistance in the set of isogenic cell lines studied.-5 M carboplatin in complete medium  once every week, the docetaxel resistant cell line A2780DXL was maintained by adding 4.05 × 10-7 M docetaxel in complete medium bi-weekly and resistance of the carboplatin/docetaxel dual resistant cell line A2780CBNDXL was maintained by treating with 6.07 × 10-6 M carboplatin and 6.07 × 10-9 M docetaxel in complete medium bi-weekly.The human ovarian carcinoma cell line A2780 was purchased from the European Collection of Cell Cultures  and maintained in RPMI-1640 medium with 2mM Glutamine, which contained 10% fetal bovine serum (FBS), and 1% Penicillin /Streptomycin solution . The A2780 ovarian cancer line is likely of the endometrioid subtype . The drug resistance of the carboplatin resistant cell line A2780CBN was maintained by adding 2.22 × 10Cells were assayed for sensitivity to carboplatin, docetaxel, and combined carboplatin/docetaxel using a clonogenic assay that quantifies the number of colonies generated from viable cells .50. Statistical analysis to determine if the IC50 of each resistant line was significantly different from the co-cultured parental control was calculated using Student’s t-test with two-tailed distribution and unequal variance. A p-value ≤0.05 indicated that a significant difference existed between the two sets of data (n = 3).The number of colonies growing was recorded by taking photomicrographs of five random fields (100X magnification) per drug concentration and counting the colonies in each field. The average for each drug concentration was normalized to the average of the drug free control to generate a survival fraction. The software program Graph Pad Prism  was used to plot a survival curve using the function “log [inhibitor] vs. normalized response with variable slope” to calculate the IC50 of the parent line and doses were increased 3.00-fold, 1.50-fold or 1.25-fold, depending on the ability of the cells to continue proliferating. For the A2780CBNDXL cell line, the concentrations of carboplatin and docetaxel were raised together by the same factor each time. A co-cultured control was developed for each resistant line, as described, to control for changes in gene expression due to continuous culture.Cell line selection was performed as described in a previous study by Guo et al. . Briefly5 cells per 9.60 cm2 in six well plates. Three wells were counted for each day of a four day growth curve analysis using a Vi-cell XR cell viability analyzer . Three biological replicates of the growth curve experiment were performed. Averages of viable cell numbers were plotted for each day and student’s t-test  was applied to determine if the average cell number per day was significantly different or not from the parental line. Population doubling time was calculated using the formula G = t * log(2)/(log(Nt) − log(N0)), where G = generation or doubling time, t = time period (hr.), Nt = number of cells at time t, N0 = initial number of cells.Cells were plated at a density of 2.0 × 10Total RNA from each cell line was prepared using RNeasy Mini Kit (50) # 74104 from Qiagen Inc.  according to the manufacturer’s instructions. RNAase OUT  was added to prevent RNA degradation. Integrity of total RNA samples was assessed using capillary electrophoresis on an Agilent 2100 Bioanalyzer. RNA samples with RIN values of 8.0 or higher were considered intact and appropriate to use for microarray analysis.Changes in gene expression between A2780 parental cells and the derived drug resistant cell lines were observed using Agilent 4 × 44 whole human genome arrays . A 500 ng aliquot of total RNA, isolated with a Qiagen RNeasy mini kit  was used for each sample. The RNA was labeled with Cy3 or Cy5 using and Agilent Quick Amp Labeling kit (Product # 5190–0444). Hybridization was performed as per the manufacturer’s protocol. Experiments were repeated using multiple batches of labeled RNA, with both forward and reverse-labeling to account for dye bias, for a total of 4 (A2780CBN and A2780CBNDXL) or 8 (A2780DXL) two-color arrays. The microarrays were scanned, and feature extraction and background intensity corrections were performed with Agilent software (v.10.7.3.1). Using a 3-way ANOVA, Partek Genomics suite  was used to generate a list of genes significantly over- or under-expressed with false discovery rates of 0.01 and 0.05, with a cut-off value of ± 2-fold change in gene expression. The microarray data was deposited in the NCBI Gene Expression Omnibus (GEO) database in accordance with MIAME standards (GSE39337). This list was further refined in our analysis by only including genes which had p≥0.05 for the comparisons between replicate arrays and reverse labeled samples. The refined gene lists were imported into Microsoft Office Excel 2003 to perform a three-way column comparison to identify genes that were unique to each cell line, shared between two lines or shared between all three of the resistant lines. Partek Genomics Suite was also used to perform principal component analysis (PCA) and hierarchical clustering analysis of the data.Three independent RNA isolations were prepared from each cell line using Qiagen RNeasy isolation kits. Reverse transcription of cDNA was performed using the Superscript First-Strand synthesis system for QPCR from Invitrogen Canada Inc. . Sybrgreen and Taq polymerase reagents for the QPCR reactions were purchased as a GoTaq QPCR Master Mix from Promega Corporation . QPCR reactions were carried out on a BioRad Dyad Disciple Peltier Thermal Cycler using a Chromo4 Real-time PCR Detector. QPCR primers were designed to specifically amplify coding transcripts , GCLC (cat. sc-100747), FLRT3 (cat. sc-82156), CDH11 (cat. sc-52352), CYP1B1 (cat. sc-32882), ANXA1 (cat. sc-12740), GAPDH (cat.sc-47724) and GSTO1 (cat. sc-130318) were purchased from Santa Cruz Biotechnology, Inc. . The antibody for MT2A (cat. H00004502-M01) was purchased from Abnova Corporation . Antibodies against AKR1C3 (Clone NP6.G6.A6) and γ-tubulin (cat. T5192) were acquired from Sigma-Aldrich Canada, Ltd. . Binding of antibodies was detected using Luminol enhanced chemoluminescence (ECL) reagents from Santa Cruz Biotechnology, Inc. and images were recorded and analyzed using an Alpha-Innotech gel documentation system with Alpha-Ease software system  or by exposing film.For the microarray data, significant differences in fold change expression between co-cultured controls and resistant lines was determined using the Partek Genomics Suite program . Significant differences between log Ct values for the parent and resistant lines, normalized to log Ct values for the S28 transcript, were determined using Student’s t-test. To determine fold change for the qPCR data, the average relative quantity of gene expression for each gene was determined using MJ Opticon Monitor Analysis Software v. 3.1 ). Following normalization to the S28 housekeeping gene, the fold changes were determined from the ratio between parental and resistant lines. ANOVA followed by Tukey’s post hoc test was calculated for qPCR and immunoblot log fold change data in the Graphpad Prism v. 5.02 .50 for carboplatin of 2.12 × 10-6 M. Therefore, selection for the carboplatin resistant line began at 1.00 × 10-9 M carboplatin, a dose in the 1000 fold range below the IC50 of the parent line, and continued until a maximally tolerated dose (MTD) of 2.22 × 10-5 M was reached. A maximally tolerated dose was considered to be achieved when cell viability dropped below 30% at the next higher dose. Clonogenic assays performed on the A2780CBN cell line at this point revealed an IC50 of 7.77 x 10-5 M to carboplatin, while the A2780 co-cultured parental control (A2780CC) had an IC50 of 5.73 × 10-6 M carboplatin. The IC50 values for the A2780CBN and A2780CC were statistically different by Student’s t-test . The ratio between the resistant and co-cultured control IC50 values demonstrated about a 13-fold increase in the IC50 of the resistant A2780CBN line  for the A2780DXL and A2780CC cell lines, establishing a lack of cross resistance to carboplatin in the A2780DXL line.A clonogenic assay was also carried on the A2780DXL cell line to test for cross resistance to carboplatin. Plotting of the data generated an ICTerminal dose cultures of the A2780CBN, A2780 DXL and A2780CBNDXL cell lines and the parental A2780 line, were plated without drug to determine the effect of selection on cell doubling time (growth rate). Fresh medium was provided to the day 3 and4 cultures on day 2. Curves representing the average proliferation of each cell line across three replicate experiments show that all resistant lines proliferate more slowly than the parental A2780 cell line  in each cell line compared to the matching co-cultured control were derived from the Partek Genomics Suite as described. The Partek Genomics list showed 3000 genes were significantly different in the A2780CBN line, 4621 genes were significant in the A2780DXL line and 4070 genes were significantly different in the A2780CBNDXL line. If a gene exhibited an opposite direction in fold change (upregulated in one line but down regulated in another) it was counted as unique in each line and shared between lines if they went in the same direction. Following the refinement of the lists as described, a total of 1096 unique changes in gene expression were observed for the A2780CBN cell line compared to the co-cultured parental control, 1273 unique changes were observed in the A2780DXL cell line and 1154 changes were specific to the dual resistant cell line , Tukey’s test was applied as the post hoc test to identify the cell line(s) that contained most of the difference. Significant differences among the resistant cell lines were not found for five genes  while two genes (ABCB1 and ANXA1) were significantly different among all three cell lines  experiments, only the AKR1C3, ANXA1, CYP1B1, GCLC, GAPDH, MT2A and γ-tubulin antibodies produced measurable immunoblot signals Figure . Followiin vitro[in vitro and characterization of cells selected for resistance to both classes of agents.In this study, a set of three isogenic drug-resistant ovarian cancer cell lines has been generated from the A2780 ovarian cancer cell line. The A2780 cell line has the advantage of being derived from a chemo-naïve patient, and is therefore sensitive to many chemotherapeutics -50 but hin vitro-55. In a50 of the parental A2780 cell line, generated populations of resistant cells and avoided selection of a few drug resistant clones. This selection strategy may not seem to reflect the typical clinical approach of treating patients with high doses delivered in several cycles, but the dose administered to a patient is not likely reflective of the amount of drug that actually reaches a tumor. Studies of intratumoral drug distribution have shown that drug concentrations vary within a tumor, that not all tumor cells may experience a lethal dose, and that other factors such as intratumoral cell heterogeneity and tumor microenvironment interactions can interfere with consistent, high dose delivery of a drug in a tumor [in vitro conditions, we believe our approach beginning with a low concentration and gradually increasing the dose is more likely to mimic the variable and gradually increasing drug environment in a tumor and to select for a population of drug resistant cells representative of the cell heterogeneity present in tumors. Using this selection method, the A2780CBN cell line was acquired with an IC50 of 7.77 × 10-5 M carboplatin (Table -5 M) [in vitro in ovarian tumor cells [During the selection for single or dual drug resistance in our study, the gradual increase in drug concentration, beginning with a dose 1000-fold below the IC a tumor -58. Althle -5 M) , indicator cells .50 of 3.61 × 10-7 M docetaxel, which was 4000 fold more resistant than the A2780CC. Although initially very toxic, once resistance had begun to develop, it was possible to increase the dose until this very high level of resistance occurred. Intraperitoneal delivery of docetaxel to patients was reported by Morgan et al. to result in mean peak plasma concentrations of 4.6-6.6 × 10-7 M docetaxel and 5.9-8.1 × 10-5 M mean peak intraperitoneal concentrations of docetaxel [50 of our A2780DXL line falls just under the lower end of the range, indicating that the A2780DXL cell line tolerance also falls in a clinically relevant range. In vitro resistance to paclitaxel in ovarian cell lines has been reported in this range, as well [The A2780DXL cell line had an ICocetaxel . Althoug as well .50 of 8.02 × 10-6 M for carboplatin and 8.02 × 10-9 M docetaxel .RIN: RNA integrity; FBS: Fetal bovine serum; PCR: Polymerase chain reaction; QPCR: Quantitative Real-Time Polymerase Chain Reaction; IMDM: Iscove’s Modified Dulbecco’s Medium; RPMI: Roswell Park Modified IMDM; A2780CC: Co-cultured parental control cells; A2780CBN: A2780 cell selected for resistance to carboplatin; A2780DXL: A2780 cells selected for resistance to docetaxel; A2780CBNDXL: A2780 cells selected for resistance to carboplatin and docetaxel; ICThe authors of this paper have no competing interests to declare. We have not received reimbursements, fees, funding, or salary from an organization that may in any way gain or lose financially from the publication of this manuscript, either now or in the future. No such organization is financing this manuscript, including the article-processing charge. We do not hold stocks or shares in any organization that may gain or lose financially from the publication of this manuscript, either now or in the future. We do not hold and are not currently applying for any patents relating to the content of the manuscript. We have not received reimbursements, fees, funding, or salary from an organization that holds or has applied for patents relating to the content of the manuscript. We have no other financial competing interests. We have no non-financial competing interests  to declare in relation to this manuscript. The author(s) declare that they have no competing interests.SRA generated the resistant cell lines, assisted in performing the microarray experiments, and performed the data subset analysis and selection of the validation genes. RN performed and analyzed the qPCR validation experiments for the selected genes. BG carried out the microarray experiments and the initial data analysis to identify genes with significantly different expression in the resistant cell lines. AP contributed to the conception of the study, reviewed the manuscript and oversaw the microarray experiments. KLM performed the proliferation analysis of the cell lines and analyzed the data. SC carried out the immunoblots and generated the graphs. CL contributed to the conception of the study, oversaw the selection of the cell lines, oversaw the proliferation analysis, contributed to the final data analysis and drafted the manuscript. All authors read and approved the final manuscript.Table S1. Primer sequences and melting temperatures. Click here for fileTable S2. Determination of significant difference between the parent and resistant cell lines according to QPCR analysis.Click here for fileFigure S1. Comparison of gene expression changes between the resistant cell lines. The log fold change in gene expression is shown for each resistant cell line. One-way ANOVA followed by Tukey’s test was performed to determine if there was any significant difference among the cell lines in the level of gene expression and if the difference could be assigned to any cell line(s). Significant difference between cell lines is indicated by lettering above the columns.Click here for file


However, it has been shown that mRNA expression profiles of these cell lines were closer to dedifferentiated in vivo thyroid tumors  than to differentiated ones. Here an overview of the knowledge of these models was made. The mutational status of six human thyroid cancer cell lines  was in line with previously reported findings for 10 genes frequently mutated in thyroid cancer. However, the presence of a BRAF mutation (T1799A: V600E) in WRO questions the use of this cell line as a model for follicular thyroid carcinoma (FTC). Next, to investigate the biological meaning of the modulated mRNAs in these cells, a pathway analysis on previously obtained mRNA profiles was performed on five cell lines. In five cell lines, the MHC class II pathway was down-regulated and in four of them, ribosome biosynthesis and translation pathways were up-regulated. mRNA expression profiles of the cell lines were also compared to those of the different types of thyroid cancers. Three datasets originating from different microarray platforms and derived from distinct laboratories were used. This meta-analysis showed a significant higher correlation between the profiles of the thyroid cancer cell lines and ATC, than to differentiated thyroid tumors  specifically for DNA replication. This already observed higher correlation was obtained here with an increased number of in vivo tumors and using different platforms. In summary, this would suggest that some papillary thyroid carcinoma or follicular thyroid carcinoma (PTC or FTC) cell lines  might have partially lost their original DNA synthesis/replication regulation mechanisms during their in vitro cell adaptation/evolution.Human thyroid cancer cell lines are the most used models for thyroid cancer studies. They must be used with detailed knowledge of their characteristics. These  Among the various possible experimental models, human cancer cell lines are frequently used  and leads to the constitutive kinase activity of the protein  and PAX8/PPARγ rearrangements (approximately 35% of the cases). Mutations of the RAS gene activate the mitogenic MAPK and the PI3K pathways. PAX8/PPARγ rearrangements are the result of a fusion between the 5′ end of the PAX8 transcription factor and the 3′ end of PPARγ, a member of the nuclear hormone receptor superfamily, constitutively expressed in thyrocytes. Different rearrangements between the 2 genes have been described, and sometimes reported within a single tumor  and the PI3KCA oncogenes (10% of the cases) have been described in this tumor type.The genetic changes most often found in FTC are TP53 and β-catenin genes.ATC is the most dedifferentiated, aggressive thyroid cancer  than to differentiated thyroid cancers (PTC and FTC) ; the TPC1 cell line from Prof. M. Mareel ; the K1 cell line from Dr. Zaruhi Poghosyan . The BCPAP, TPC1 and 8505C cell lines were cultured in RPMI 1640 (w L-glutamate) completed by foetal bovine serum (10%), penicillin/streptomycin (2%) and amphotericine B (1%); the FTC133, WRO and K1 cell lines were cultured in Dulbecco's Modified Eagle Medium, Nutrient mixture F-12  completed by foetal bovine serum (10%), penicillin/streptomycin (2%), amphotericine B (1%). All culture reagents were purchased from Gibco .After removal of the culture medium, 2 ml of TRIzol Reagent  was added to the cells grown in 10 cm dishes. Total RNA was extracted according to the manufacturer's instructions, followed by purification on miRNeasy columns . RNA was spectrophotometrically quantified, and its integrity was verified by automated gel electrophoresis .2 (50 mM), 1 μl dNTP mix , 1 μl of the forward and the reverse primer (10 μM each), 0.4 μl Taq DNA polymerase (5U/μl), 2 μl DNA from the RT reaction and 38.1 μl of water. Primers and PCR conditions are detailed in Table After a DNase treatment with DNase I Amplification Grade kit , 1 μg of total RNA was used for reverse transcription using hexamers [(3.6 μg/μl) ] and reverse transcriptase . The PCR reactions were performed with the recombining Taq DNA polymerase kit (Invitrogen). Each PCR reaction was performed in the presence of 5 μl 10X PCR buffer, 1.5 μl MgClhttp://blast.ncbi.nlm.nih.gov/Blast.cgi).PCR products were purified with the QIAquick PCR purification kit (QIAGEN) according to the manufacturer's instructions. Sequencing was performed with the BigDye Terminator V3.1 Cycle Sequencing Kit  with the sequencer ABI PRISM 3130 (Applied Biosystems) and the genetic analysis program 3130-XI. Sequences were analyzed with BLAST  from NCBI (www.ulb.ac.be//medecine/iribhm/microarray/data/)  was <5%. The analysis was performed separately for up-regulated and down-regulated genes.In order to investigate the biological meaning of the modulated mRNAs, Database for Annotation, Visualization and Integrated Discovery  . Based on the merged file, correlation coefficients between gene expression profiles of ATC and thyroid cancer cell lines were compared to correlation coefficients between gene expression profiles of PTC or FTC and thyroid cancer cell lines, using a Mann–Whitney q-values were computed based on a slightly modified version of Rank Products . Similarly, genes that had significant regulations in opposite directions between ATC and cell lines were further analyzed.Scores and BRAF, NRAS, HRAS, KRAS, TP53, PI3KCA, PTEN genes was explored as well as the presence of RET/PTC1, RET/PTC3, and PAX8/PPARγ rearrangements. Therefore, primers were designed to target the point mutations and the rearrangements most frequently found in thyroid carcinomas  mutation and K1 the heterozygous PIK3CA G1624A (E542K) mutation, in addition to the BRAF mutation  were further analysed  mutation was detected although these cells were originally described as BRAF wild-type  was confirmed in accordance with previously published data . By using data derived from different microarray platforms and from two different laboratories, thereby including a larger number of in vivo tumors, the current data confirm our previous finding that mRNA expression profiles from human thyroid tumor cell lines with different origins are the closest to undifferentiated in vivo tumors  might have partially lost their original DNA synthesis/replication regulation mechanisms during their in vitro cell adaptation/evolution. Also, further comparisons using microarray data set from poorly differentiated thyroid cancers are needed and could be a near future goal in our laboratory.In this work, the aim was to further investigate the characteristics of human thyroid cancer cell lines derived from different origins. First, genetic alterations in six cell lines were in line with literature data and furthermore, the BRAF mutation in WRO cells questions this cell line as a model for FTC, indeed this mutation does not occur in FTC. Second, in five cell lines the MHC class II pathway was down-regulated and in four of them, ribosome biosynthesis and translation pathways were up-regulated. Third, by using microarray data from ATC, PTC, and FTC, we have preliminary found that mRNA expression profiles of human thyroid cancer cell lines, originally derived from ATC, poorly differentiated PTC, FTC, or PTC, were closer to ATC. Specifically, we found genes commonly up-regulated in ATC involved in DNA replication which were also up-regulated in the thyroid cancer cell lines, in accordance with their higher proliferation rate During our study, the origin of the WRO cell line as thyroid cancer cell line has been questioned. However, the pathway analysis and the comparisons of expression profiles showed similar results for WRO cell line and the other thyroid cancer cell lines. To address the origin of the WRO cell line, more complete analyses (including cell lines from different cancer types) could be a near future goal in our laboratory.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.


V600 mutant melanoma is limited primarily by the development of acquired resistance leading to tumor progression. Clinical trials are in progress using MEK inhibitors following disease progression in patients receiving BRAF inhibitors. However, the PI3K/AKT pathway can also induce resistance to the inhibitors of MAPK pathway.The sustained clinical activity of the BRAF inhibitor vemurafenib (PLX4032/RG7204) in patients with BRAFThe sensitivity to vemurafenib or the MEK inhibitor AZD6244 was tested in sensitive and resistant human melanoma cell lines exploring differences in activation-associated phosphorylation levels of major signaling molecules, leading to the testing of co-inhibition of the AKT/mTOR pathway genetically and pharmacologically. There was a high degree of cross-resistance to vemurafenib and AZD6244, except in two vemurafenib-resistant cell lines that acquired a secondary mutation in NRAS. In other cell lines, acquired resistance to both drugs was associated with persistence or increase in activity of AKT pathway. siRNA-mediated gene silencing and combination therapy with an AKT inhibitor or rapamycin partially or completely reversed the resistance.in vitro models results in frequent cross resistance to MEK inhibitors, except when the resistance is the result of a secondary NRAS mutation. Resistance to BRAF or MEK inhibitors is associated with the induction or persistence of activity within the AKT pathway in the presence of these drugs. This resistance can be potentially reversed by the combination of a RAF or MEK inhibitor with an AKT or mTOR inhibitor. These combinations should be available for clinical testing in patients progressing on BRAF inhibitors.Primary and acquired resistance to vemurafenib in these  V600E is a dominant activating mutation in melanoma resulting in a constitutive activation of the mitogen-activated protein kinase (MAPK) pathway and uncontrolled cell growth V600E mutant metastatic melanoma treated with the type I RAF inhibitor vemurafenib (previously know as PLX4032 or RG7204) V600E mutation have all the features of oncogene addiction BRAFV600E mutant melanoma Similar to other cancers, melanomas have frequent alterations in the phosphatidylinositol 3-kinases (PI3K) and v-akt murine thymoma viral oncogene homolog 1 (AKT) pathway, another key signal transduction pathway governing cell growth and survival. The most common alterations are genomic or functional loss of PTEN and amplification and point mutations in AKT V600E mutant melanomas may continue to depend on the MAPK even after progressing on BRAF inhibitors, through the reactivation of phosphorylated ERK in resistant cells V600E mutant melanoma had a response but then progressed on BRAF inhibitors like vemurafenib or dabrafenib.Analysis of clinical samples provided evidence that the antitumor activity of vemurafenib is mediated by inhibition of ERK signaling in vitro exposure, and those established from patient-derived biopsies progressing after vemurafenib. Recent studies have shown that in addition to the dependence on MAPK pathway, over expression of receptors such as the platelet-derived growth factor beta (PDGFRβ) or the insulin growth factor-1 receptor (IGF-1R), which are upstream of PI3K/AKT pathway, may play important roles in the resistance to BRAF inhibitors V600E mutant melanomas not responding to vemurafenib are also cross-resistant to single agent MEK inhibitors, but co-targeting of the AKT/mTOR pathway provides means of treating most of these resistant cells.In this study we first tested the concept of treating with a MEK inhibitor upon progression on a BRAF inhibitor in selected melanoma cell lines that encompassed cell lines with primarily resistance to vemurafenib, those with acquired resistance to vemurafenib after Vemurafenib  was obtained under a materials transfer agreement (MTA) with Plexxikon  and Roche . It was dissolved in DMSO  to a stock concentration of 100 mM. AZD6244 was purchased from Selleck Chemicals  and dissolved in DMSO to a 100 mM stock. The isozyme-selective AKTi VIII  was dissolved in DMSO to a stock concentration of 10 µM. Rapamycin in EtOH  was used for the drug combination experiments at the noted concentrations. Human melanoma cell lines (M series) were established from patient's biopsies under UCLA IRB approval #02-08-067 as previously described 2- and water-saturated incubator. The medium was changed when most cells had attached and cell cultures were then propagated and passaged in vitro as needed. These in vivo resistant cultures were not routinely selected with constant exposure to vemurafenib.Four cell lines were derived from patients participating in the phase I (NCT00405587) or phase II (NCT00949702) clinical trials with vemurafenib as previously described in vitro acquired resistance, BRAFV600E mutant melanoma cell lines with in vitro sensitivity to vemurafenib were plated in T25 flasks and treated with their respective IC50 of vemurafenib as described previously 50 for the parental cell line. The subline was labeled as the parental cell line followed by “AR” for acquired resistance.To generate cell lines with Melanoma cell lines were treated in triplicate with vemurafenib, AZD6244, AKTi or rapamycin (or the combinations) and parallel vehicle control in the given concentrations for 72 hours. Cell viability was measured using a tetrazolium compound [3--5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based colorimetric cell proliferation assay  as previously described Information for the oncogenic characterization of the cell lines tested herein was based mainly on data we have previously reported 5 cells suspended in 1.25 ml of the culturing media. Then, each sample was plated for protein isolation after 72 hours or RNA isolation after 48 hours or for the drug treatment after 24 hours and assessment of proliferation rate after 120 hours.Cell lines were transfected with the gene-specific or no target control siRNAs . To perform the transfection, Lipofectamine™ RNAiMAX reagent  was used according to the Reverse Transfection method described by the manufacturer. In brief, 40 pmol of each siRNA was diluted separately into 250 µl of serum free phenol red free RPMI 1640. To each diluted siRNA, 3 µl of Lipofectamine™ RNAiMAX was added. Each transfecting mixture was added to 1.8×10Western blotting was performed as previously described To perform the apoptosis assay cells were treated with the solvent (DMSO) or 2 µM of vemurafenib, AZD6244, AKTi or 10 nM of rapamycin or combination of these compounds. After 48 hours of treatment, protein lysated were prepared from each condition and analyzed by Western blotting method for the detection of cleaved caspase-3.5′CTGAGCGGAACATTCTAGAGT 3′ and Reverse 5′-AAGTCGGTCAGTTTGATGTGG-3′) were used to amplify a cDNA fragment of ∼300 base pares. The primers span the sides of an intron and do not amplify the genomic DNA. As a control for the integrity and level of RNA, GAPDH was amplified by specific Forward (5′-GTCAACGGATTTGGTCGTATT 3′) and Reverse (5′-AGTCTTCTGGGTGGCAGTGAT 3′) primer set. RT-PCR cycles were as follows: Reverse transcription at 50°C for 30 minute, 95°C for 2 minutes, 27 cycles of 94°C (15 Sec), 56.5°C (30 Sec), 68°C (30 Sec), and 68°C for 5 minutes. The samples were analyzed on 1.5% agarose gel.RT-PCR assay was used to investigate the efficiency of ribosomal protein S6 kinase, 2 (RPS6KB2 or here in S6K2) knockdown by siRNA cocktail at mRNA level. M238 and M238AR2 cell lines were transfected with a combination of 25 pmol of S6K2 siRNA plus 25 pmol of S6K1 siRNA or with 50 pmol of control SiRNA (as described in the above). Cells were harvested after 48 hours and total RNA was isolated by using RiboPure™ Kit . RT-PCR assays were performed on 350 ng of total RNA isolated from each sample by using SuperScript III One-Step RT-PCR System with Platinum® Taq kit . Specific S6K2 set of primers  to calculate the combination index (CI). A CI equal to 1 indicates an additive effect; a CI less than 1 indicates synergy. With the use of CalcuSyn software, synergy is further refined as synergism (CI = 0.3–0.7), strong synergism (CI = 0.1–0.3), and very strong synergism (CI<0.1).To determine synergistic, additive, or antagonistic effects of the drug combinations, we used the combination index method of Chou and Talalay 50 s above 10 µM for each agent were considered resistant. M229, M238 and M249 are three BRAFV600E mutant parental cell lines sensitive to vemurafenib in vitro culture in increasing concentrations of vemurafenib also exhibited a complete  or a partial (M249-AR4) resistance to single agent AZD6244. The other three cell lines  obtained from metastatic melanoma lesions surgically resected from patients progressing on vemurafenib had different sensitivities to both agents. M370 was established from an intra-cardiac mass which developed six months after starting on vemurafenib. It corresponds to the sample labeled as Pt48 R in ref. in vitro sensitivity to vemurafenib but was resistant to AZD6244. M376 was established from a lymph node metastatic lesion that partially regressed on vemurafenib but increased in size 10 months after starting the therapy. It corresponds to the sample labeled as Pt55 R in ref. in vitro sensitivity to both vemurafenib and AZD6244. For these studies, the cell lines obtained from patients treated with vemurafenib had been cultured ex vivo without the presence of low dose vemurafenib for several passages, which is different from studies performed by Nazarian et al.V600E mutant cell lines with primary resistance to vemurafenib ex vivo but completely resistant to AZD6244. Thus, with the exception of M249-AR4 and M376, BRAFV600E mutant melanoma cell lines resistant to vemurafenib have cross resistance to the MEK inhibitor AZD6244.We analyzed if melanoma cell lines resistant to vemurafenib would be sensitive to the MEK inhibitor AZD6244 using MTS assays . Cell liQ61K, in addition to the BRAFV600E mutation, in the in vitro acquired resistant cell line M249-AR4 and in the patient-derived acquired resistant cell line M376, was associated with resistance to vemurafenib but sensitivity to AZD6244, corroborating the earlier study Q61K-induced signaling while the MEK inhibitor AZD6244 blocks the pathway, and consequently inhibits the cell proliferation in these cell lines. Secondary mutations in BRAF, KRAS, NRAS or HRAS were not noted in any other cell line We explored the oncogenic alterations in this panel of cell lines using targeted oncogene sequencing and SNP arrays . The prein vitro acquired resistant sublines and four of the patient-derived cell lines to explore signaling pathways that may be differentially modulated upon treatment with the RAF or MEK inhibitors for 24 hours , as well as in M376 which was sensitive. p-ERK1/2 levels were lower in all the AZD6244 treated samples regardless of their sensitivity to either one of the drugs.We selected two pairs of parental and their 24 hours . Vemurafin vitro acquired resistant cells M238-AR2 and M229-AR9, and the patient-derived resistant cell lines. Both vemurafenib and AZD6244 caused decreases in p-S6 levels in sensitive cell lines but almost no clear changes in the their resistant sublines. However, it seemed that the decrease in p-S6 was not associated with the pattern of response to the drugs except when accompanied by a decrease in p-p70 S6K1. 4-EBP-1 is another important factor downstream of AKT/mTOR pathway that has been reported to link this pathway to the MAPK pathway The differences in p-AKT/p-p70 S6K1/p-S6 pathways were more pronounced between sensitive and resistant cell lines. Vemurafinib and AZD6244 induced similar changes in p-AKT levels. However, it seemed that these changes were cell line-dependent, showing simultaneous increases in p-AKT T308 and S473 (suggesting feedback) in the resistant cell lines M238-AR2, M229-AR9 and M370, while there were no obvious changes in M395, and decreases in both p-AKT levels in M238 and M380. Thus, none of the cell lines highly resistant to vemurafenib showed decreases in one or both p-AKT levels after the exposure to the drugs. In fact, vemurafenib (and to a lesser extent AZD6244) treatment of vemurafenib-acquired resistant cell lines consistently increased p-AKT levels, with the notable exception of the N-RAS mutated line, M376. Exposure of sensitive cell lines, M229 and M238, to even low concentrations of the drugs caused drastic decreases in their p-p70 S6K1 and p-S6 levels. However, particularly at lower concentrations of the drugs, there was less or no effect on p-p70 S6K1 levels in the mTORC complexes have been described as the central node for regulation of growth and metabolism. RICTOR is one of the proteins in mTORC2 complex that phosphorylates AKT at Ser473 through a feedback mechanism. This phosphorylation is necessary for the complete activation of AKT. p70 S6K1 is one of the key factors downstream of mTOR that phosphorylate and induces S6 activity. Given the pattern of p-AKT induction primarily in the non-NRAS mutant acquired resistant melanoma cell lines we focused on the effects of inhibiting the mTORC complexes or p70 S6K1. In M238-AR2 and its parental cell line, transient knockdown of each target was achieved by transfection of cells with the target specific siRNAs pool that resulted in a reproducible knockdown of at least 70% at protein levels . A highlAs it was expected, knockdown of RICTOR caused an evident decrease in phosphorylation of p-AKT at Ser473. Interestingly, knockdown of RICTOR also caused a clear decrease in phosphorylation of 4-EBP-1 particularly in the presence of vemurafenib or AZD6244, and also a detectable decrease in p-S6 level only in M238-AR2 treated with the agents . These rRAPTOR is one of the main proteins in mTORC1 complex. In our experiments, knockdown of RAPTOR caused detectable decreases in phosphorylation of p-p70 S6K1 in both parental and resistant cell line. However, knockdown of RAPTOR in these cell lines caused no detectable decrease in p-S6 level . KnockdoTo examine clinically-relevant means of addressing primary or acquired resistance to single agent MAPK inhibitors we tested the addition of an AKT1/2 or a mTORC1 pharmacological inhibitor. All cell lines were resistant to single agent AKT inhibitor (AKTi) or rapamycin  and 5. Ain vitro sensitive/adaptive resistant pair cell lines, we detected cleaved caspase-3 (CC3) levels by Western blotting method Click here for additional data file.Figure S2Effects of RAPTOR knockdown by siRNAs in combination with either vemurafenib or AZD6244. The efficiency of siRNA knockdowns and its effect on downstream signaling determined by Western blot analysis of protein lysates (a). M238 parental (b) and M238-AR2 resistant subline (c) were transfected with RAPTOR siRNAs and cultured in increasing concentrations of vemurafenib or AZD6244. The effect of raptor knockdown on resistance and growth inhibition was analyzed after 120 hours by an MTS assay. D in each graph refers to the un-transfected untreated cells and is used as the 100% reference point for all the conditions in the assays.(TIF)Click here for additional data file.Figure S3Cleaved caspase-3 in sensitive and adaptive resistant cell lines treated with vemurafenib, AZD6244, rapamycin, AKTi. Cell lines were treated by the solvent (DMSO), 2 µM of vemurafenib, AZD6244, AKTi or 10 nM of rapamycin for 48 hours. Each sample was analyzed by Western blotting using a cleaved caspase-3 (CC3) specific antibody.(TIF)Click here for additional data file.Figure S4Diagram of pathways and possible cross-talk points involved in survival and resistance of melanoma cell lines.(TIF)Click here for additional data file.


The cell cycle, the influence of cobalt chloride-induced hypoxia-inducible factor-1α (HIF-1α) and the glucose transporter 1 (GLUT-1) expression  were analysed after ascorbate exposure under normoxic and hypoxic conditions. The amount of ascorbyl radicals increased with rising serum concentrations. Hypoxia (0.1% O2) globally increased the IC50 of ascorbate in the 60 cancer cell lines from 4.5 ± 3.6 mM to 10.1 ± 5.9 mM , thus inducing cellular resistance towards ascorbate. This ascorbate resistance depended on HIF-1α-signalling, but did not correlate with cell line-specific expression of the ascorbate transporter GLUT-1. However, under normoxic and hypoxic conditions, ascorbate treatment at the individual IC50 reduced the expression of GLUT-1 in the cancer cells. Our data show a ROS-induced, HIF-1α-and O2-dependent cytotoxicity of ascorbate on 60 different cancer cells. This suggests that for clinical application, cancer patients should additionally be oxygenized to increase the cytotoxic efficacy of ascorbate.Intravenous application of high-dose ascorbate is used in complementary palliative medicine to treat cancer patients. Pharmacological doses of ascorbate in the mM range induce cytotoxicity in cancer cells mediated by reactive oxygen species (ROS), namely hydrogen peroxide and ascorbyl radicals. However, little is known about intrinsic or extrinsic factors modulating this ascorbate-mediated cytotoxicity. Under normoxia and hypoxia, ascorbate IC The cell lines of the NCI60 panel were chosen for this study as (i) they are well-defined and well-characterized , (ii) they are the standard tool used for cytotoxicity testing in the screening of novel drugs by the NCI and (iii) as they consist of different tumour entities, thus enabling a detailed comparison.Regarding the currently observed limited efficacy of high-dose ascorbate on the killing of tumour cells in cancer patients in clinical phase I trials 2)-induced HIF1α e.g. via HBO treatment) of the patient to assure for adequate tissue oxygen levels necessary for ascorbate-driven ROS generation. This might improve the therapeutic anti-cancer properties of high-dose ascorbate in the cancer patient.Our results indicate that the generation of ROS from ascorbate is catalysed by serum and that severe hypoxia (as present in cancer metastases) mediates a reduced susceptibility (or resistance) of all 60 cancer cell lines towards ascorbate-induced inhibition of cell proliferation (driven by induction of apoptosis). This ascorbate resistance was enhanced by cobalt chloride (CoCl3) on a Bruker ESP300E x-band spectrometer  equipped with a TM 4103 resonator operating at 9.8 GHz. Instrument settings were as follows: modulation frequency 100 kHz; microwave power 5 mW; modulation amplitude, gain and time constant varied within the range of ascorbyl radicals. Electron spin resonance spectroscopy measurements were performed in triplicates.To detect the induction of ascorbyl radicals by ascorbate, ESR was applied. Ascorbyl radical induction was measured in medium with 8 mM ascorbate. Electron spin resonance spectroscopy ascorbyl spectra were recorded as described previously 2O2), nine different cell lines were exposed to 8 or 16 mM ascorbate or to 0.5 mM H2O2, with or without the addition of 100 μg/ml catalase in RPMI full medium (refer to Cells and cell culture for details) for 1 hr. The medium was discarded, cells were washed once with PBS and H2O2 was measured in PBS by using dichlorofluorescein substrate  according to the procedure described by the supplier. Experiments were performed in quadruplicates.For the generation of peroxide radicals  was reduced to 0.1% O2 .All 60 cell lines . Ascorbate or CoCl2 was added directly to the culture medium of the cells. Cells treated with culture medium only served as controls. 100 μM CoCl2 was applied for 24 hrs before ascorbate treatment or harvesting the cells to induce HIF1α.The following chemicals were used: Injectable vitamin C solution , CoCl50 concentration for each cell line. The following concentrations of ascorbate were applied to determine the IC50 value: 32, 16, 8, 4, 2 and 1 mM, 500, 250, 125, 62.5, and 31.25 μM. Cells exposed to culture medium or culture medium only served as controls. All experiments were performed in quadruplicates. The exact IC50 value was calculated by using GraphPad Prism 5 (Nonlinear Regression EC50 shift). The assay was started following incubation for 24 hrs. Medium was discarded, each well was washed twice with PBS (without Ca2+ and Mg2+) and 100 μl of a solution containing 100 mg 4-methylumbelliferyl heptanoate per ml PBS was added. Plates were incubated at 37°C for 1 hr and measured in a Fluoroskan II  with an λem of 355 nm and an λex of 460 nm. The intensity of fluorescence indicates the number of viable cells in the wells Cells were seeded as triplicates in 96-well plates at a density of 2500 cells per well in 50 μl medium. After 24 hrs, medium was replaced by medium containing ascorbate in rising concentrations to determine the IC50 concentrations (as determined by the proliferation assay described above). After 24 hrs, cells (1 × 106) were harvested, washed with cold PBS, fixed with 70% ethanol and incubated at 4°C over night. Cells were then centrifuged and washed twice in cold PBS. Intracellular DNA was labelled with propidium iodide solution [propidium iodide 40 mg/ml (Sigma-Aldrich) and RNase 100 mg/ml  in PBS] and incubated at 4°C for 30 min. in the dark. Cell cycle was analysed by using flow cytometry and FACSDiva software . 100,000 cells were analysed per treatment group.Cells were treated with ascorbate at their respective IC50 concentrations, and cells were further incubated for 24 hrs. Cells were next washed with PBS and lysed for 20 min. on ice with a lysis buffer . Cell lysates were cleared by centrifugation for 5 min. at 13.000 g at 4°C and 20 μg protein subjected to SDS–PAGE and transferred to polyvinylidene difluoride membranes. After blocking in PBS/0.1% Tween-20/5% dry milk for 1 hr at RT, the blots were incubated overnight with the primary antibodies directed against anti-GLUT-1 , anti-HIF1α  and against anti-β-actin , in PBS/0.1% Tween-20/5% dry milk, washed with PBS for 3 × 10 min. and incubated with secondary HRP-conjugated antibody . After washing with PBS for 3 × 10 min., the ECLplus detection reagent  was used for detection of. The membrane was immersed in ECL solution for 1 min. and then exposed to X-ray film . For the detection of HIF1α, a CCD-camera was used . For densitometric analysis of the Western blot bands, we used ImageJ software or LabWorks analysis software (Ultra-Violet Products Ltd). GLUT-1 and HIF1α protein bands were normalized to the respective actin band.Cells were seeded into T75 flasks and incubated for 24 hrs. Medium was replaced by medium containing ascorbate at ICt-test and Mann–Whitney t-test. P < 0.05 was considered statistically significant. Fitted midpoints of the two dose–response curves (logEC50) under normoxia and hypoxia were statistically compared by using the F-test function for non-linear curve fits of GraphPad Prism 5 and P < 0.05 was considered significant. Two-way anova was used for the statistical multiple comparisons of ascorbate-treated cells in combination with hypoxia and/or CoCl2 treatment.Statistical analyses were performed with a two-tailed unpaired −•; Fig. −•  resulted in an increase in H2O2 formation in all of the cancer cell lines  independent of the tumour type; as expected, the addition of 100 μg/ml catalase completely destroyed the ascorbate-induced H2O2 in all nine cell lines  by using ESR. The addition of ascorbate (8 mM) to the medium yielded a detectable amount of ascorbyl radicals . The results of two exemplary cell lines (OVCAR-4 and NCI-H23) are displayed in Figure 50 concentration was calculated. These two consecutive steps were performed for all 60 cell lines. We found a dose-dependent inhibition of cell proliferation in all cell lines, independent of their mutation status (Table S1) 24 hrs after treatment with pharmacological concentrations achievable in humans via i.v. administration of >10 g ascorbate e.g. lung and prostate cancer cell lines; Fig. 50 value of ascorbate for all 60 cancer cell lines under normoxic conditions was 4.5 ± 3.6 mM. For the different tumour types, we found the following IC50 values of ascorbate: renal cancer cell lines (n = 8): 5.2 ± 3.1 mM; breast cancer cell lines (n = 6): 2.4 ± 1.7 mM; prostate cancer cell lines (n = 2): 13.2 ± 11.1 mM; colon cancer cell lines (n = 7): 3.7 ± 3.8 mM; lung cancer cell lines (n = 9): 5.9 ± 3.6 mM; leukaemia cell lines (n = 6): 0.6 ± 0.7 mM; melanoma cell lines (n = 9): 3.1 ± 2.8 mM; ovarian cancer cell lines (n = 7): 3.7 ± 3.2 mM; glioblastoma cell lines (n = 6): 3.0 ± 1.3 mM  to determine the cytostatic efficacy (inhibition of cell proliferation) for each cell line under normoxic conditions , the influence of severe hypoxia (0.1% O2) on ascorbate-induced decrease in proliferation was analysed. We chose 0.1% O2 treatment for 24 hrs to mimic chronic hypoxia as recently described for prostate cancer cells 50 value increased for every cell line ; breast cancer: 6.8 ± 5.4 mM, 2.8-fold increase ; prostate cancer: 22.9 ± 17.5 mM, 1.7-fold increase ; colon cancer: 8.6 ± 5.6 mM, 2.3-fold increase ; lung cancer: 12.2 ± 8.0 mM, 2.1-fold increase ; leukaemia: 1.2 ± 1.3 mM, 2.0-fold increase ; melanoma: 9.7 ± 9.7 mM, 3.1-fold increase ; ovarian cancer: 7.9 ± 7.5 mM, 2.1-fold increase ; glioblastoma: 8.8 ± 0.8 mM, 2.9-fold increase . The following cell lines did not reach a statistically significant increase of the respective IC50 under hypoxia: HT578T, HT-29, HCC2998, HCT-15, CCRF, NCI-H23, NCI-H460, HOP-62, NCI-H322M, NCI-H226, M14, SK-MEL-5, OVCAR-8, PC-3, A498, RXF-393, ACHN.Simultaneously to the determination of the ICditions 2% O2, the2-dependent cytotoxic effects of ascorbate were dependent on the hypoxia-inducible factor-1α signalling cascade or simply on O2-availability, we applied CoCl2, a HIF1α-inducing drug 50 concentration of ascorbate under hypoxia  or both on cancer cell viability after ascorbate exposure at increasing concentrations  for 1 hr. Hypoxia rendered all of the cells resistant towards ascorbate-induced cell death  significantly increased the ascorbate resistance in all of the six tested cancer cells. As expected, the addition of catalase blocked the ascorbate-induced proliferation inhibition  cell-cycle analyses were performed on all 60 cancer cell lines after incubation with ascorbate at the individual IC50 concentration for 24 hrs under normoxic (21% O2) and hypoxic (0.1% O2) conditions. The percentage of cells in the sub-G1 phase (indicating apoptotic cells) after ascorbate incubation varied between the different cell lines  was applied in each case. Figure 50 concentration of each of the 60 cancer cell lines are given in Table S2. Together, the FACS analyses demonstrate that pharmacological ascorbate primarily induced an increase in the sub-G1 cell population under normoxic conditions in 47 of 60 cells lines (Table S2). Under hypoxic conditions, we observed a comparable result after additional ascorbate treatment: In 45 of 60 cell lines, the sub-G1 cell population was increased. As expected . However, our results yielded cell type-specific differences in GLUT-1 expression after ascorbate treatment and under hypoxia , the cellular GLUT-1 protein levels increased in 40 of the 60 cell lines. This effect occurred in most of the melanoma and breast cancer cells. This is in line with the fact that GLUT-1 is a downstream-target of HIF-1α, which is induced by hypoxia e.g. in the non-small cell lung cancer cell lines). Under normoxic conditions, ascorbate treatment at the individual IC50 concentrations decreased GLUT-1 expression in 30 of the 60 cell lines, with the most pronounced effects in melanoma, non-small cell lung cancer and breast cancer cell lines  mediated a reduced susceptibility towards ascorbate-induced inhibition of cancer cell proliferation. This ascorbate resistance was enhanced by CoCl2-induced HIF1α. However, the ascorbate-induced cytotoxicity did not correlate with the endogenous GLUT-1 expression.The clinical experience from phase I trials shows that in cancer patients, high-dose ascorbate bears only little cytotoxic efficacy 50 values for ascorbate were determined for all 60 cell lines under normoxic (21% O2) and hypoxic (0.1% O2) conditions. Under normoxic conditions, all cell lines had IC50 values that were in the range of concentrations that can be achieved in patients by i.v. administration of ascorbate 50 values. This might account for the fact that in pilot clinical trials on stage IV cancer patients by using high-dose pharmacological ascorbate, the cytotoxic efficacy was lower 2 partial pressure  or whether HIF1α-signalling might also be involved in the observed cellular resistance. We used CoCl2 as inductor for HIF1α 2 partial pressure (hypoxia) and the expression of HIF1α drove resistance towards ascorbate-induced cell death. To determine the mode of action of ascorbate-induced inhibition of cancer cell proliferation in more detail, we performed FACS cell-cycle analyses on all 60 cell lines after incubation with ascorbate at the individual IC50 under normoxic and hypoxic conditions. The FACS analyses yielded an increased sub-G1 (apoptotic) population in the majority of the cell lines after incubation with ascorbate under normoxic conditions, which was significantly reduced under hypoxic conditions. The high percentage of cells in the sub-G1 phase after incubation with ascorbate indicated that the induction of apoptosis was the major antiproliferative mode of action of ascorbate in cancer cells. However, in some of the cell lines analysed, no apoptosis induction was observed despite the obvious growth inhibition after incubation at the respective IC50 concentration. In such cases, pyknosis/necrosis probably caused the cytotoxic effects In a first approach, the individual ICin vitro. This cytotoxicity is executed by ascorbyl radicals and H2O2 and is catalysed by serum components. Hypoxic conditions and HIF-1α-signalling, both present in cancer metastases, confer resistance to the cancer cells towards ascorbate-induced cytotoxicity, possibly via up-regulation of pro-survival HIF-1α downstream-targets (e.g. GLUT-1). At the same time, under hypoxic conditions, pharmacological ascorbate treatment partly reduces the expression of GLUT-1. These findings might be the key for the lack of clinical efficacy of high-dose ascorbate in cancer patients up to this day. We conclude that in future clinical trials, the respective patients should be oxygenized in addition to ascorbate therapy to break drug resistance.Our results demonstrate that pharmacological doses of ascorbate bear cytotoxic effects on the NCI60 panel of cancer cells