Molecular alterations of the MAPK pathway are frequently observed in papillary thyroid carcinomas (PTCs). It leads to a constitutive activation of the signalling pathway through an increase in MEK and ERK phosphorylation. ERK is negatively feedback-regulated by Dual Specificity Phosphatases (DUSPs), especially two ERK-specific DUSPs, DUSP5 (nuclear) and DUSP6 (cytosolic). These negative MAPK regulators may play a role in thyroid carcinogenesis.V12 or BRAFV600E). Expression levels of DUSP5 and DUSP6 were quantified in 39 human PTCs. The functional role of DUSP5 and DUSP6 was investigated through their silencing in two human BRAFV600E carcinoma cell lines.MAPK pathway activation was analyzed in 11 human thyroid cancer cell lines. Both phosphatases were studied in three PCCL3 rat thyroid cell lines that express doxycycline inducible PTC oncogenes (RET/PTC3, H-RASV600E human thyroid cancer cell lines expressed higher phospho-MEK levels but not higher phospho-ERK levels. DUSP5 and DUSP6 are specifically induced by the MEK-ERK pathway in the three PTC oncogenes inducible thyroid cell lines. This negative feedback loop explains the tight regulation of p-ERK levels. DUSP5 and DUSP6 mRNA are overexpressed in human PTCs, especially in BRAFV600E mutated PTCs. DUSP5 and/or DUSP6 siRNA inactivation did not affect proliferation in two BRAFV600E mutated cell lines, which may be explained by a compensatory increase in other phosphatases. In the light of this, we observed a marked DUSP6 upregulation upon DUSP5 inactivation. Despite this, DUSP5 and DUSP6 positively control cell migration and invasion.BRAFV600E PTCs. DUSP5 and DUSP6 have pro-tumorigenic properties in two BRAFV600E PTC cell line models.Our results are in favor of a stronger activation of the MAPK pathway in BRAF In papillary thyroid carcinoma (PTC), the most common thyroid malignancy, activating-mutations of genes encoding effectors of the mitogen-activated protein kinase (MAPK) pathway are central for malignant transformation. This pathway transduces mitogenic signals via activation of receptor tyrosine kinases, leading to the successive recruitment and activation of RAS and members of the RAF family of serine/threonine kinases. A cascade of phosphorylation is initiated, culminating in the activation by phosphorylation of MEK and consequently of the extracellular signal-regulated kinases (ERK).RET and activating-point mutations of RAS or BRAF are found in 70% of all cases of PTC and are mutually exclusive. Mutation of BRAF, especially a valine to glutamic acid substitution at codon 600 (V600E) termed the BRAFV600E mutation, is the most frequent genetic alteration, identified in 60% of cases (range: 27%-87%) [BRAF activating mutation is closely associated with extrathyroidal extension, lymph node metastasis, advanced tumor stages, disease recurrence, and even patient mortality [BRAF mutation with worse prognosis compared to PTC with no or other genetic alterations has so far only been partially elucidated. There is evidence that the transcriptional output of the MAPK pathway is greatest in BRAFV600E mutant tumors, because this oncoprotein signals as a monomer and is therefore unresponsive to feedback inhibition on RAF dimers [Rearrangements of the gene encoding the receptor tyrosine kinase 27%-87%) , 2. PTCsF dimers .Levels of ERK phosphorylation (p-ERK) are dictated by the coordinated activities of protein kinases and phosphatases. Identical p-ERK immunostaining levels described in human PTC regardless of the genetic alteration , 5 couldDUSP6 is a candidate tumor suppressor gene, especially in pancreatic cancers, which have a high prevalence of KRAS mutations [DUSP5 and DUSP6 are overexpressed [V600E) [utations . In PTCsxpressed , 12. DUS [V600E) . Althoug [V600E) . The funV600E thyroid cancer cell lines, thus suggesting a pro-tumorigenic role of these phosphatases in PTC.We hypothesized that DUSP5 and DUSP6 may be biomarkers of the MAPK output in PTCs, in the context of a negative feedback loop. Alternatively, they could act as tumor suppressor genes. For that purpose, we studied the MAPK pathway activation, the expression and regulation of DUSP5 and DUSP6, and the consequences of their inactivation in thyroid cancer cell lines. We found out that DUSP5 and DUSP6 are overexpressed in human thyroid carcinomas and are surrogate markers of MAPK pathway activation. Silencing of DUSP5 or DUSP6, or both phosphatases in thyroid cell lines does not affect proliferation, possibly explained by compensation between phosphatases of the DUSP family. Finally, DUSP5 and DUSP6 silencing reduced the cell migration and invasion capacities of two BRAFV12 and BRAFV600E are three cells lines derived from the well differentiated, non-transformed rat thyroid cell line PCCL3 that conditionally express one of the three known thyroid oncogenes , in a doxycycline-dependent manner [PCCL3 RET/PTC3, HRASt manner –16. Thest manner .We used a panel of 11 human thyroid cancer cell lines. The 8505c (DSMZ number: ACC 219) and B-CPAP (DSMZ number: ACC 273) cell lines were purchased from the German Collection of Microorganisms and Cell Culture. This cell bank genetically fingerprinted these last two cell lines by multiplex PCR of minisatellite markers that revealed a unique DNA profile. All other cell lines were provided by J. A. Fagin  in 2010 and used with permission from: N.-E. Heldin : HTh74 , C643 1, SW1736 ® cDNA Reverse Transcription Kit .Formalin fixed paraffin-embedded (FFPE) PTC and adjacent normal thyroid specimens were obtained from the Departments of Pathology at the Cochin and Ambroise-Paré University Hospitals in Paris, France. Informed signed consent for genetic diagnosis, tumor analysis, and for access to the data collected was obtained from all the patients. Total RNA was extracted from a 10-μm section of the FFPE tumor tissue using TRIzol Reagent  according to the manufacturer’s protocol and 1μg was reverse transcribed to generate cDNA using High-CapacityBRAF gene and exon 1 and 2 of the HRAS, NRAS, and KRAS genes were amplified. Direct sequencing of the purified fragments was then performed using the Genetic Sequencer ABI3100 Applied Biosystems apparatus. Sequence comparisons were carried out using the Chromas Program.Exon 14 and 15 of the ® 480 Real-Time PCR System  as already described [For RET/PTC rearrangements, unbalanced expression of exons 12 and 13 relative to exons 10 and 11 of RET were searched, by quantitative real-time PCR (qPCR) using LightCyclerescribed . Then, tDUSP5 and DUSP6 was calculated for all tumors using the ΔΔCt method and GAPDH as an internal control.Relative expression of The MEK inhibitor, UO126  and selumetinib AZD6244  and the PI3K inhibitor, LY294002  were used with the indicated concentration.™, Roche) and protease inhibitor cocktails . Proteins were quantified using the Bradford protein assay and separated by SDS-polyacrilamide gel (10%), then transferred onto nitrocellulose membrane.Cells were lysed in a buffer containing 20 mmol/L Tris-HCl (pH 7.5), 1mmol/L EDTA, NaCl 150 mM, NP40 1%) phosphatase , mouse monoclonal anti-p-ERK , rabbit polyclonal anti-p-MEK 1/2 , rabbit polyclonal anti-total-ERK , rabbit polyclonal anti-total-MEK1 , rabbit polyclonal anti-total-MEK2 , mouse monoclonal anti-DUSP6  and rabbit polyclonal anti-GAPDH . Primary antibody against DUSP5 was provided by Stephen Keyse [The following primary antibodies were used: mouse monoclonal anti-BRAF® cDNA Reverse Transcription Kit (Applied Biosystems). LightCycler® 480 Real-Time PCR System (Roche) was used for qPCR. The following primer pairs were used: DUSP5 (5’-GCC CGC GGG TCT ACT TCC TCA AA-3’ and 5’-ATT TCA ACT GGG CCA CCC TGG-3’), DUSP6 (5’- TCC CTG AGG CCA TTT CTT TCA TAG ATG-3’ and 5’-GCA GCT GAC CCA TGA AGT TGA AGT-3’), GAPDH (5’- GCC ACA TCG CTC AGA CAC CA -3’ and 5’- TTC CCG TTC TCA GCC TTG AC -3’) and cyclophilin (5’- ATGGCACTGGTGGCAAGTCC-3’ and 5’-TTGCCATTCCTGGACCCAAA-3’). Relative expression of DUSP5 and DUSP6 was calculated using the ΔΔCt method and the cyclophilin or GAPDH as an internal control.Total RNA (500 ng to 1μg) extracted from PCCL3 cells and human cell lines by RNeasy Mini Kit  was reverse transcribed to generate cDNA using High-CapacityDUSP5 (siDUSP5 (1): 5’-GGCCTTCGATTACATCAAG- 3’, siDUSP5 (2): 5’-GAGAAGATTGAGAGTGAGA- 3’, siDUSP5 (3); 5’- GGAAGUGCCUACCAUGCAU- 3’) for human DUSP6 (siDUSP6 (1): 5’-GAACTGTGGTGTCTTGGTA-3’ siDUSP6 (2): 5’- GGAATTCGGCATCAAGTAC- 3’), and a control siRNA (5’- GGCATAGATGTAGCTGTAA- 3’) were purchased from Eurofins MWG Operon. siRNAs were transfected at the concentration of 100 nmol/L with Lipofectamine Plus . After transfection, the suppression of targeted proteins was determined by reverse transcription qPCR and Western blotting. Cell viability was determined as described below.For small interfering RNA (siRNA) experiments, siRNA for human Thiazolyl Blue Tetrazolium Bromide (MTT) (Sigma-Aldrich) was dissolved in PBS at 5 mg/ml and filtered. Cells were seeded in 96-well plates and incubated at 37°C. Stock MTT (5mg/ml) solution (10 μ1 per 100μ1 medium) was added to all wells (10 wells per condition in each experiment) of an assay. After 4 hours of additional incubation, 100 μl of 0.04 N HCI in isopropanol was added to each well. Plates were then read within 1 hour on an automatic enzyme linked immunosorbent assay plate reader . The optical density was calculated as the difference between the absorbance at a reference wavelength (690 nm) and that at a test wavelength (570nm) corrected to the blanks. Within each experiment, the means and the standard errors of the means were calculated.Forty-eight hours after transfection with DUSP5 or DUSP6 siRNA, cells were suspended in 1% foetal calf serum (FCS) DMEM medium into the upper chamber of 24-well transwell chambers . Previously DMEM medium containing 10% FCS were added to the lower chambers. For the invasion assay, the transwell membranes were coated with Growth Factor Reduced Matrigel (12.5 μg in 60 ml/well) (BD Biosciences) and dried for 1 h. Cells were transferred onto the artificial basement membrane.After overnight incubation at 37°C, cells that invaded the Matrigel layer and/or migrated to the lower chamber were fixed in methanol, stained with Giemsa and counted under an inverted microscope. Assays were performed in triplicate, cells were either counted in adjacent fields (n = 9) and data were reported as average cell number per field or with an Image J software macro and data were reported as arbitrary density unit.3 8505C cells or 30.103 BCPAP cells) were seeded on 6-well plates in 0.3% agar in culture medium over a base layer of 0.7% agar, Colonies were microscopically counted after 3 weeks of culture.The anchorage-independent growth was analyzed through a soft agar colony formation assay. Twenty four hours after transfection with siRNA, cells  [Wilcoxon and paired t tests were used to compare s (TCGA)  Reverse s (TCGA) . UnpaireV600E mutated cell lines, as confirmed by Western Blot with a sensitive and specific anti-BRAFV600E antibody (V600E mutated cell lines (V600E mutated cell lines in comparison with others (p = 0.007), suggesting a higher MAPK pathway activation by BRAFV600E. The same results were obtained with serum-starved (i.e. 0.5% FBS) cells  cells .V600E mutated cell lines.We did not find a significant correlation between MAPK activation i.e. phospho-MEK levels and DUSPs expression at the mRNA and protein levels in our 11 human thyroid cancer cell lines . HoweverSince p-ERK levels tend to be the same regardless of the genetic alteration, we assumed that the steady state of p-ERK levels would be maintained by high levels of feedback inhibitors that dephosphorylate ERK and allow tight regulation of p-ERK levels. Accordingly, we studied the regulation of DUSP5 and DUSP6, known to specifically dephosphorylate and inactivate ERK.V12 or BRAFV600E), in a doxycycline-dependent manner. We first demonstrated that a strong induction of oncogenic RET/PTC3, HRASV12 or BRAFV600E was correlated with a marked increase in DUSP5 and DUSP6 mRNA levels compared to baseline during a 72 hours kinetic of doxycycline treatment   and LY294002 (PI3K inhibitor), we tested the involvement of the ERK1/2 and PI3K/Akt pathways respectively, in the up-regulation of tivation . DUSP5 arylation . On the m of MEK . This waAZD6244) . On the 0E cells .DUSP5 and DUSP6 induction was blocked by MEK inhibition, using a highly potent and selective MEK inhibitor namely AZD6244 [Furthermore we confirmed in our five BRAF-mutated human thyroid carcinoma cell lines that  AZD6244  Fig 3)..DUSP5 anDUSP5 and DUSP6. We hypothesized that DUSP5 and DUSP6 mRNA levels might be a marker for MAPK pathway activation, with differences according to the genetic alteration present in the carcinoma. To test this hypothesis, DUSPs expression were quantified by qPCR in a total of 39 human PTCs, characterized for known MAPK pathway genetic alterations  and ten PTCs with no identified genetic alteration) and their normal adjacent thyroid tissue. DUSPs mRNA levels were found overexpressed in PTCs compared to normal adjacent thyroid tissue, with an average ratio of 1.43 for DUSP5 (p = 0.001) and 2.26 for DUSP6 (p = 0.001). DUSP5 and DUSP6 mRNA are statistically overexpressed (p = 0.007 and 0.01 respectively) in BRAF-mutated tumors in comparison with all tumors harboring either RET/PTC rearrangement or RAS mutation or none of these genetic alterations (P = 6.3.10−58) and 1.5 fold higher in RAS-mutated PTC than in normal adjacent tissue (P = 8.10−3). DUSP6 mRNA levels were 6.7 fold higher in BRAF-mutated PTC than in normal adjacent tissue (P = 3.3.10−87) and 2.2 fold higher in RAS-mutated PTC than in normal adjacent tissue (P = 1.6.10−10).We showed in the PCCL3 cell lines that the MAPK pathway drives the expression of erations . To go fTo determine if DUSPs overexpression in BRAF mutated PTCs correlates with a MAPK pathway overactivation, we performed a bioinformatic analysis of the recently published Reverse Phase Protein Lysate Microarray (RPPA) data from TCGA. We compV600E mutated human thyroid carcinoma cell lines (BCPAP and 8505c). Each siRNA effectively suppressed the targeted gene at the mRNA and protein level, but did not result in significant inhibition of cell viability  than in BRAF wild-type PTCs, with the use of a highly sensitive technique, i.e. Reverse Phase Protein Lysate Microarray. Previous immunohistochemistry (IHC) data in human PTCs did not show significant differences in the levels of p-MEK between BRAF mutated and BRAF wild type PTCs, maybe reflecting the lack of sensitivity of IHC-based detection techniques for very small differences [In this paper, higher levels of p-MEK in human BRAFoncogene . These iferences , 34.V600E PCCL3 cells during the kinetic confirmed the concept that the pathway is insensitive to upstream feedback inhibition. The observation that MEK kinase inhibition does not further induce p-MEK in BRAFV600E PCCL3 and human thyroid cancer cells further supports this concept. On the opposite, p-MEK levels remained low during the kinetic in RET/PTC3 cells reflecting multiple negative feedback mechanisms upstream of MEK and accordingly, p-MEK levels were further induced after treatment with the MEK kinase inhibitor as well as in BRAF wild-type human thyroid cancer cells (release of negative feedback loop from ERK). Altogether, these results suggest that in vitro MEK phosphorylation but not ERK phosphorylation is a better marker of MAPK pathway output, as already shown in melanoma [The increasing p-MEK levels in BRAFmelanoma . Despitemelanoma .Increased MAPK pathway activation leads to an increased feedback inhibition at multiple levels of the kinase cascade to prevent overactivation of signaling output. As p-ERK levels were relatively constant from one human or rat thyroid cell lines to another, we sought to study DUSP5 and DUSP6 which act respectively in the nucleus and the cytoplasm to dephosphorylate ERK, to maintain its steady state levels.DUSP5 and DUSP6 expression is regulated by the MEK-ERK pathway but not the PI3K pathway in PCCL3 cells and BRAF-mutated human PTC cell lines. DUSP5 and DUSP6 overexpression in PTCs has been shown by gene expression profile studies [DUSP5 and DUSP6 are overexpressed in PTCs compared to normal adjacent thyroid tissue. BRAFV600E mutated PTCs expressed significantly higher levels of these phosphatases in comparison with other PTCs suggesting higher MAPK pathway activation, which is consistent with the recently published TCGA data [DUSP5 and DUSP6 expression are markers of elevated output of the MAPK pathway.We confirmed using two MEK inhibitors (UO126 and AZD6244) that CGA data . TherefoV600E mutation status [Immunohistochemistry-based technique detection demonstrated that DUSP6 levels are increased in PTCs in comparison with benign thyroid neoplasms. Nevertheless, those levels were not correlated with BRAFn status . The comn status , 37–40 in status , 40. Morn status . As a con status , which mDUSP5 and DUSP6 reported in thyroid cancers [in vitro in human gastric and uterine cervix cancer cells and was responsible respectively for enhanced cell proliferation or massive apoptosis [In vivo, mice lacking DUSP5 show a greatly increased sensitivity to mutant Ras-driven papilloma formation in a pharmacological model of induced skin carcinogenesis [V600E and RET/PTC human thyroid cancer cell lines [RET/PTC1 rearrangement. We report here for the first time the positive role of DUSP5 on in vitro cell migration and invasion in two BRAFV600E thyroid tumor cell lines, as indicated by the decreased capacity of migration and invasion after DUSP5 invalidation in the 8505c and BCPAP cell lines. We found that DUSP6 silencing clearly reduced the invasion and migration ability of 8505c cells, in line with the results of Degl’Innocenti et al. who found reduced neoplastic properties of BRAFV600E NIM-1 thyroid carcinoma cells after DUSP6 silencing [It could be hypothesized that both phosphatases play a crucial role in tumorigenesis through the fine-tuning of magnitude and duration of ERK activation . Absence cancers  as well  cancers  support poptosis , 43. In ogenesis . In thisll lines , 31. Howll lines . Complexll lines . While Dll lines , on the ilencing . While wilencing , we founilencing . We alsoDUSP1 and DUSP2 [DUSP5 and DUSP6 mRNA levels are markers of activation of the MAPK signaling pathway in PTCs. Higher output of the MAPK pathway in BRAFV600E mutated thyroid carcinomas provides an explanation for their poorer prognosis compared to other PTCs. DUSP5 and DUSP6 have no tumor suppressor properties in two BRAFV600E thyroid carcinoma models, but instead they seem to have a protumorigenic role on thyroid carcinogenesis. The lack of effect in proliferation after specific inhibition of DUSP5 or DUSP6 or both DUSPs suggests redundancy and functional compensation in the large family of DUSP. Future work should focus on transcriptomic analysis of DUSPs expression modification, more specifically compensatory changes when one or several DUSPs are inactivated in human thyroid cancer cell lines.Pronounced compensatory increase in transcription of one DUSP when the other is knockdowned has been observed for nd DUSP2 . In our nd DUSP2 . This innd DUSP2  and are,S1 FigRET/PTC rearrangement, two with a RAS activating point mutation, and five with the BRAFV600E mutation. Protein expression levels were assayed by immunoblot for the BRAFV600E mutation, phosphorylated MEK (p-MEK), total MEK1 (t-MEK1), phosphorylated ERK (p-ERK), total ERK (t-ERK) and GAPDH.Eleven human thyroid cancer-derived cell lines were grown in 0.5% FBS medium for 48h and studied for MEK and ERK activation: two cell lines without known MAPK pathway genetic alteration, one with a (TIF)Click here for additional data file.S2 FigDUSP5 and DUSP6 mRNA were then analyzed using real time Reverse-Transcription qPCR and normalized to cyclophilin mRNA levels. DUSP5 and DUSP6 mRNA levels in BCPAP cells were arbitrary set at 1. DUSP6 mRNA levels in TTA1 cells were almost undetectable. B. Whole cell lysates of the eleven human thyroid carcinoma cell lines were subjected to immunoblotting with DUSP5 and DUSP6 antibodies.Cells were maintained for 48h in 0.5% FBS medium. A (TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 FigA. BRAF-mutated and BRAF-wild type human thyroid cancer cells were cultured in 0.5% FBS containing medium for 48h and then treated for 6 hours with the MEK inhibitor AZD6244. Whole cell extracts were subjected to the indicated antibodies. B. For each cell line the ratio pMEK/tMEK after AZD6244 treatment relative to the basal pMEK/tMEK ratio is represented.(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.


In silico methods of phenotypic screening are necessary to reduce the time and cost of the experimental in vivo screening of anticancer agents through dozens of millions of natural and synthetic chemical compounds. We used the previously developed PASS (Prediction of Activity Spectra for Substances) algorithm to create and validate the classification SAR models for predicting the cytotoxicity of chemicals against different types of human cell lines using ChEMBL experimental data. A training set from 59,882 structures of compounds was created based on the experimental data  from ChEMBL. The average accuracy of prediction (AUC) calculated by leave-one-out and a 20-fold cross-validation procedure during the training was 0.930 and 0.927 for 278 cancer cell lines, respectively, and 0.948 and 0.947 for cytotoxicity prediction for 27 normal cell lines, respectively. Using the given SAR models, we developed a freely available web-service for cell-line cytotoxicity profile prediction (CLC-Pred: Cell-Line Cytotoxicity Predictor) based on the following structural formula: http://way2drug.com/Cell-line/. Notwithstanding the progress in cell-based screening technology, experimental in vivo screening of anticancer drug-candidates through dozens of millions of natural and synthetic chemical compounds is rather expensive and time-consuming [in vitro and in silico tools were proposed to reduce the cost of such screening and to reveal possible mechanisms of the growth inhibition and killing of tumour cells [Oncology diseases are one of the main causes of death in the world . Despiteonsuming . Differeur cells . The stuur cells . NCI60, ur cells . Althougur cells . Since tur cells , and theur cells . Despite(Q)SAR approach—analysis of “structure-cytotoxicity” relationships –14;Connectivity map approach—comparison of drug-induced gene signatures given on tumour cells –19;Network pharmacology approach—analysis of known drug-target interactions –23;Machine learning techniques for revealing relations between cell line cytotoxicity  and microarray data –27.Despite the variety in the proposed methods and successful cases of their application, all of them require significant supplementary intellectual and technological efforts or additional experimental studies . The approaches related to the analysis of microarray data cannot be used for the virtual screening of compounds that have not been synthesized yet and that have no microarray data. Moreover, the mean accuracy (AUC) of the CMap approach validated on an independent set is approximately 0.61 for antineoplastic drugs . The mosThe recent development of freely available recourses, such as ChEMBL  and PubCPASS provides simultaneous predictions for many types of biological activities (activity spectrum) based on the structure of drug-like compounds –35. In PThe molecular structure is represented by the set of unique sub-structural atom-centric Multilevel Neighbourhoods of Atoms (MNA) descriptors of the first and second levels. These descriptors are a linear notation of atom-centred fragments in the structure of an organic molecule . They arEach atom in a molecule must be presented by an atomic symbol from the periodic table. Symbols of unspecified atom A, Q, *, or R group labels are not allowed.Each bond in a molecule must be a covalent bond represented by single, double or triple bond types only.The structure must include three or more carbon atoms.The structure must include only one component.The structure must be neutralized.The absolute molecular weight of a compound must be less than 1250.Since MNA descriptors do not represent the stereochemical peculiarities of the molecule, the substances with stereochemically different structures are formally considered to be equivalent.A leave-one-out cross-validation for all predictable types of biological activity and all substances in the PASS training set provides an estimate of the PASS prediction accuracy during the training procedure. The accuracy criterion ROC AUC (the Area Under the ROC Curve) is used. It is the estimate of the probability that positive and negative examples (active and inactive compounds) that are arbitrarily chosen from a validation set may be classified correctly by the prediction.Pa and «to be inactive» Pi. The list of predicted activities is arranged in descending order according to Pa–Pi values. Thus, the more probable activity types are at the top of the list. If the user chooses a higher value of Pa as a cut-off for selection of probable activities, the chance to confirm the predicted activities by the experiment is also high, but many existing activities will be lost. For instance, if Pa>0.5 is used as a threshold, about half of the real activities will be lost; for Pa>0.7, the portion of lost activities is 70%, etc.The predicted activity spectrum in PASS is represented by a list of activities with probabilities «to be active» Pa and Pi are measures that belong to both subsets of "active" and "inactive" compounds and the probabilities of the 1st and 2nd types of prediction error, respectively. These two interpretations of probabilities Pa and Pi are equivalent and can be used for interpreting the results of prediction. They can also be used for the construction of different criteria to predict the results of the analysis corresponding to the specific practical tasks.By definition, the probabilities http://dev.mysql.com/) was used. The script for the generation of the training sets was written in PHP language. ChEMBL_23 contained data for more than 1.7 million compounds, with information regarding their structures and interactions with over 11.5 thousand targets, including human tumour and normal cell lines. Two training sets were created from the ChEMBL data. One of the training sets contained the data on chemical cytotoxicity against human tumour cell lines, and the one was for human normal cell lines. The names of the cell-lines were used as in ChEMBL to provide links to the experimental data. The data from ChEMBL and Cellosaurus were used to distinguish cancer cell lines from non-cancer ones. Database of Cross-Contaminated or Misidentified Cell Lines was used to find in ChEMBL and to exclude from our training set misidentified cell lines where no authentic stock was ever found [The ChEMBL database was used as a resource for cytotoxicity data of chemicals . The dater found .Structure Data File (SDF) format was used to save the extracted information. Single small molecular-weight organic compounds with electroneutral structures were selected during the creation of the training sets. The IG50 , IC50  and % inhibition (of activity) values were analysed. The compounds were considered active if the IG50 and IC50 values were less than 10000 nM or if the percent of inhibition was higher than 50%. All compounds were considered inactive for the appropriate cell line if they were not active for this cell line according to the above-mentioned criteria. The selected cell lines contained at least 3 active and 10 inactive compounds. All the records of compounds that were simultaneously classified as active and inactive for the appropriate cell line were excluded.The training set of 59,882 unique structures of compounds was created based on the experimental data from the ChEMBL (version 23), which reflects the current knowledge about the cytotoxic substances to 943 human cell lines. This training set was used to train PASS for the creation of classification models of “structure-cytotoxicity” relationships. Only cell lines for which the cytotoxicity was predicted with the accuracy of prediction (AUC) higher than 0.8 were selected. The average accuracy of prediction calculated by a leave-one-out cross-validation (LOO CV) procedure was 0.948 for the cytotoxicity prediction for 27 normal cell lines  and was The AUC range for the different cell lines was from 0.800 (DMS-114—lung carcinoma) to 1.000 . The most accurate prediction among organs with several cell lines was obtained for four head and neck cell lines , two upper aerodigestive tract cell lines , and eight liver cell lines . The colon, breast, and lung cell lines also showed a mean accuracy of prediction of AUC > 0.9 , but no strict correlation was found between the number of substances in the training set or the number of cell lines in the category and accuracy of prediction.http://www.way2drug.com/Cell-line/). The example of the prediction of cell line cytotoxicity for Sorafenib  is shown in A freely available web-service PASS CLC Pred for the prediction of cytotoxicity of chemicals against tumour and normal human cell lines from different tissues was created based on the abovementioned PASS models  Integrity Database .The prediction results, which are shown in the Supplements , were cohttp://www.way2drug.com/gusar/acutoxpredict.html), which predicts the LD50 values for chemicals with oral, intravenous, intraperitoneal and subcutaneous routes of administration [http://www.way2drug.com/PASSOnline/) service may be used for predicting the possible molecular mechanisms of action related to the cytotoxic action of compounds [The ChEMBL database provides freely available experimental data on the cytotoxicity study of compounds against more than 1500 cell lines. These data allowed us to create reasonable classification SAR models for predicting the cytotoxicity of chemicals against 305 cell lines. Most of these cell lines (278) are different types of tumours related to the organs or tissue, which are of great interest in the development of new drugs or the repositioning of known drugs. ChEMBL includes much less information about the cytotoxicity of compounds against normal cell lines. Therefore, the number of classification models for the prediction of cytotoxicity of chemicals against normal cell lines is less. The models were created only for 27 normal cell-lines, which also belong to different organs or tissues. The ability to predict the cytotoxic action of compounds against normal cell lines is very important for estimating the safety of drug-candidates because many of the cytotoxic compounds that are used against tumour cell lines are often cytotoxic for normal cells and must be excluded from drug-candidates. The prediction of the cytotoxicity of chemicals against normal cell lines may be helpful for estimating the safety of drug-candidates for development in other therapeutic fields of application. The estimation of the general toxicity of compounds at the level of the organism may be given by our previously developed freely available web service for the prediction of rat acute toxicity  should increase the threshold of the Pa value. Together with the rules of interpretation of the prediction results described in the materials and methods, it should be noted that prediction results including the cytotoxicity against several cell lines from the same organ or tissue increase the probability of finding cytotoxic compounds acting on tumours located in that organ or tissue. The web service started working in 2016. Since then, several studies to assess the cytotoxicity of natural compounds have been conducted by independent researchers –48.in silico or in vitro interactions with antineoplastic targets may not reveal the general cytotoxic effect on cell lines. Revealing the cell effects depends not only on the interaction with the targets but also on many other parameters, e.g., interaction with transporters, passage through a cell membrane, and regulation of signal and metabolic pathways, which depend on cell-line-specific cancer mutations and changes in gene expression. Therefore, the combination of the computational estimation of the cytotoxic effect of chemicals in different cell-lines together with the estimation of the ligand-target interactions provides a more effective method for the design of new antineoplastic drugs. An example of the application of such approach was recently provided in our publication on the search for new compounds with cytotoxicity against breast cancer cell lines [The traditional single target or multi-target based drug designs aim at estimating the interaction between the ligand and target(s). In this paradigm, some potential drug-candidates that display ll lines .S1 Fig(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 File(PHP)Click here for additional data file.S2 File(CSV)Click here for additional data file.S3 File(CSV)Click here for additional data file.


The present study is to discover a new genes associated with drug resistance development in ovarian cancer.We used microarray analysis to determine alterations in the level of expression of genes in cisplatin- (CisPt), doxorubicin- (Dox), topotecan- (Top), and paclitaxel- (Pac) resistant variants of W1 and A2780 ovarian cancer cell lines. Immunohistochemistry assay was used to determine protein expression in ovarian cancer patients.We observed alterations in the expression of 22 genes that were common to all three cell lines that were resistant to the same cytostatic drug. The level of expression of 13 genes was upregulated and that of nine genes was downregulated. In the CisPt-resistant cell line, we observed downregulated expression of ABCC6, BST2, ERAP2 and MCTP1; in the Pac-resistant cell line, we observe upregulated expression of ABCB1, EPHA7 and RUNDC3B and downregulated expression of LIPG, MCTP1, NSBP1, PCDH9, PTPRK and SEMA3A. The expression levels of three genes, ABCB1, ABCB4 and IFI16, were upregulated in the Dox-resistant cell lines. In the Top-resistant cell lines, we observed increased expression levels of ABCG2, HERC5, IFIH1, MYOT, S100A3, SAMD4A, SPP1 and TGFBI and decreased expression levels of MCTP1 and PTPRK. The expression of EPHA7, IFI16, SPP1 and TGFBI was confirmed at protein level in analyzed ovarian cancer patients..The expression profiles of the investigated cell lines indicated that new candidate genes are related to the development of resistance to the cytostatic drugs that are used in first- and second-line chemotherapy of ovarian cancer. Ovarian cancer is one of the most lethal gynecological malignancies. Most patients are diagnosed with an advanced disease and have a poor prognosis . At the The most significant mechanism underlying the resistance to cytostatic drugs is their active removal from cancer cells by drug transporters of the ABC family . These tThe first-line chemotherapy for advanced ovarian cancer always involves a platinum-based drug [carboplatin or cisplatin (CisPt)] and a taxane [paclitaxel (Pac) or docetaxel] . The secCisPt is the most frequently used antitumor agent and the most important drug used in ovarian cancer chemotherapy. This drug reacts with the nitrogen atoms of DNA and preferentially reacts with the N-7 atom of deoxyguanylic acid. This process results in intrastrand and interstrand DNA cross-linking, consequently inhibiting DNA synthesis and transcription . ResistaThe second most important drug for ovarian cancer treatment is Pac. Pac belongs to the family of antimitotic anticancer agents and blocks mitosis through stabilizing microtubules, leading to the blockage of cell division and thus, cell survival . The mosThe most important drugs used in the second-line chemotherapy of ovarian cancer are Dox and Top. Top and Dox are inhibitors of DNA topoisomerase I and II, respectively. Topoisomerases are enzymes that regulate the overwinding or underwinding of the DNA helix . TopoisoHowever, it sometimes is difficult to explain the development of drug resistance in cancer cells based on the patterns of expression of the genes that are known to be involved in drug resistance, which indicates that yet-unknown genes and mechanisms are also involved in drug resistance.In this study, we compared the gene-expression profiles of ovarian cancer cell lines resistant to the cytostatic drugs used in first-line chemotherapy, CisPt and Pac, and those used in second-line chemotherapy, Dox and Top. Drug-resistant cell lines were derived from primary (W1) or established (A2780) ovarian cancer cell lines. Alterations in the levels of gene expression of the drug-resistant cell lines were determined using oligonucleotide microarrays.The quality of all of the GeneChip expression data was within the “good sample” limits according to the values for the parameters evaluated in the analysis of the preliminary data, such as the background and noise averages, percentage of present calls, presence of internal hybridization controls as increasing signals, presence of poly-A controls as decreasing signals and the GAPDH to β-actin 3′/5′ signal ratios.The genes that were associated with Cis, Dox, Pac or Top resistance were selected. The contribution to drug resistance of the genes for which the expression level was significantly changed in the drug-resistant cells relative to that in their drug-sensitive counterparts by greater than 5-fold and less than 0.2-fold  was evaluated. Genes with expression levels of between 5- and 0.2-fold those of the controls were considered ‘not significant (NS)' when the gene lists were constructed.Analysis of the gene expression of ovarian cancer cell lines resistant to each cytostatic drug provided information about the response of the cancer cells to the different cytostatic drugs during treatment. Figure In addition to the four genes that were underexpressed in all three of the Cis-resistant cell lines, six genes were overexpressed and 13 genes were underexpressed in both the A2780CR1 and A2780CR2 cell lines.In addition to the three genes that were overexpressed and the six genes that were underexpressed in all three of the Pac-resistant cell lines, 14 genes were overexpressed and eight genes were underexpressed in both the A2780PR1 and A2780PR2 cell lines. Seven genes were overexpressed and one gene was underexpressed in both the A2780PR1 and W1PR cell lines and three genes were overexpressed and six genes were underexpressed in both the A2780PR2 and W1PR cell lines.The expression of only three genes was changed (overexpressed) in all three of the Dox-resistant cell lines. The same seven genes were overexpressed and other set of the same seven genes were underexpressed in the A2780DR1 and A2780DR2 cell lines. One gene was overexpressed in both the A2780DR1 and W1DR cell lines and one gene was underexpressed in both the A2780DR2 and W1DR cell lines.In all three of the Top-resistant cell lines. we observed the upregulated expression of eight genes and the downregulated expression of two genes. Upregulated expression of 14 genes and downregulated expression of seven genes was observed in both the A2780TR1 and A2780TR2 cell lines. One gene was overexpressed and one gene was underexpressed in both the A2780TR1 and W1TR cell lines, and the expression of five genes was upregulated in both the A2780TR2 and W1TR cell lines.For more detailed analysis, we selected genes with expression that was up/down-regulated more than/less than 5 and -5, respectively, in three cell lines that were resistant to the same cytostatic drug. Table Collective, the expression levels of 22 genes were changed in three cell lines resistant to the same cytostatic drug. The expression of 13 genes was upregulated and that of nine genes was downregulated. We did not observe the overexpression of any gene in the cell lines resistant to CisPt. In contrast, we observed the underexpression of four genes in the cell lines resistant to this drug, which were the ABCC6, BST2, ERAP2 and MCTP1. In the Pac-resistant cell lines, we observed upregulated expression of three genes, ABCB1, EPHA7 and RUNDC3B. Six genes were underexpressed in the Pac-resistant cell lines LIPG, MCTP1, NSBP1, PCDH9, PTPRK and SEMA3A. The Dox-resistant cell lines were characterized by the increased expression of three genes, ABCB1, ABCB4 and IFI16. No gene was underexpressed in all three of the Dox-resistant cell lines. We observed the upregulated expression of eight genes in the Top-resistant cell lines ABCG2, HERC5, IFIH1, MYOT, S100A3, SAMD4A, SPP1 and TGFBI. The expression of two genes, MCTP1 and PTPRK, were downregulated in all three of the Top-resistant cell lines.The expression level of some genes had changed in response to more than one cytostatic drug. The expression of ABCB1 was increased in all of the Pac- and Dox-resistant cell lines. The expression of EPHA7 was increased in three Pac-resistant cell lines and in one Dox- and one Top-resistant cell line. IFI16 expression was increased in all of the Dox-resistant cell lines but was also increased more than four-hundred-fold in the A2780PR1 Pac-resistant cell line. We observed the increased expression of IFIH1 in three Top-resistant cell lines and in the A2780DR1 and A2780PR1 cell lines that were resistant to Dox and Pac, respectively. MYOT expression was increased in three Top-resistant cell lines and two Pac-resistant cell lines. RUNDC3B expression was increased not only in all of the Pac-resistant cell lines but also in two of the Dox-resistant cell lines. S100A3 expression was increased in all of the Top-resistant cell lines and in two of the CisPt-resistant cell lines. SAMD4A expression was increased in three Top- and two Pac-resistant cell lines. SPP1 expression was very significantly increased in all of the Top-resistant cell lines and one CisPt-resistant cell line and was less significantly increased in two Dox-resistant cell lines and one Pac-resistant cell line. The level of expression of MCTP1 was decreased in all of the CisPt-, Pac- and Top-resistant cell lines and also in one Dox-resistant cell line. The expression of PTPRK was decreased in all of the Pac- and Top-resistant cell lines and in two of the three CisPt-resistant cell lines. The expression of PCDH9 was downregulated in three Pac-resistant cell lines, two Dox-resistant cell lines and in one CisPt- and one Top-resistant cell line.Of the 22 genes for which the expression levels were analysed, the expression of three genes, ABCB1, ABCG2 and SPP1, was very significantly upregulated – by more than 50-fold – in three cell lines that were resistant to the same cytostatic drug. We also observed very greatly increased expression – by more 50-fold – of ABCB4, IFI16 and RUNDC3B, although this expression pattern was not shared by all of the cell lines resistant to the same cytostatic drug. The genes with the most highly downregulated expression – by more than 50-fold – were BST2, PTPRK and SEMA3A.Immunohistochemical analysis of selected  proteins was tested in ovarian cancer patients. The aim of this study was to verify whether the altered expression of analyzed genes and proteins observed in cell lines can be confirmed in a real cancer patient tissues as well.The positive expression of IFI16 was observed in ovarian serous adenocarcinoma patients but not in ovarian endometrioid adenocarcinoma ones Figure . Strong EPHA7 protein was detected in both ovarian serous adenocarcinoma and endometrioid adenocarcinoma cancer cells Figure . In bothModerate (2) positive reaction was observed for SPP1 and TGFBI proteins Figure . Both prIn this study alterations in the level of gene expression were found in ovarian cancer cell lines that were resistant to cytostatic drugs of first and second lines of chemotherapy. The genes with expression levels that were between 5- and 0.2-fold that of the control cells were considered not to be significantly affected, and the relationship between their expression levels and drug resistance will not be discussed.The microarray data were confirmed by the qPCR results for genes representative of the MDR phenotype (MDR1 and MDR3) (data not shown). Additionally, the results of western blotting analysis of P-gp and BCRP protein expression also correlated with the alterations in the expression levels of the genes encoding these proteins (data not shown).To our knowledge, the results presented here are the first to shown changes in the expression of genes in ovarian cancer cell lines that were resistant to cytostatic drugs of the first and second lines of chemotherapy. The levels of gene expression were determined in three resistant cell lines to make the results more credible. Our results indicated that new genes are related to the resistance to chemotherapeutic drugs.First, we selected the genes that were overexpressed/underexpressed by at least 5-fold in the drug-resistant cell lines relative to the levels in the control cells Figure . To obtaCisPt is the most important drug used to treat ovarian cancer  as well ABCC6 is drug transporter from the ABC family that transports glutathione S-conjugated leukotriene. The role of ABCC6 in drug resistance is not well established. Increased expression of ABCC6 has been reported to impart a low level of resistance to etoposide, teniposide, doxorubicin, and daunorubicin . The rolBone-marrow stromal antigen 2 (BST2), also known as a tetherin, is a protein that is associated with lipid rafts and is expressed mainly on B cells . ExpressERAP2 (endoplasmic reticulum aminopeptidase 2) plays a central role in peptide trimming, a process required for the generation of most HLA class I-binding peptides . To date2+ binding , Pac [A2780PR1 and A2780PR2 (A2780 paclitaxel resistant)], Dox [A2780DR1 and A2780DR2 (A2780 doxorubicin resistant)] and Top [A2780TR1 and A2780TR2 (A2780 topotecan resistant)] were generated by exposing A2780 cells to the relevant drugs at incrementally increased concentrations.The human ovarian cancer W1 cell line was established using ovarian cancer tissue obtained from an untreated patient. W1 sublines resistant to CisPt [W1CR (W1 cisplatin resistant)], Dox [W1DR (W1 doxorubicin resistant)], Top [W1TR (W1 topotecan resistant)] and Pac [W1PR (W1 paclitaxel resistant)] were obtained by exposing W1 cells to the drugs at incrementally increased concentrations.2 in 25 cm2 flask in dedicated media supplemented with appropriate drug. The established concentrations of the initial drugs exposure were of: CisPt 20 ng/mL, Dox 10 ng/mL, Top 0,5 ng/mL, and Pac 1 ng/mL. Each cell line was exposed three times for 3-day periods during a 3–6-week period allowing for growth recovery between cycles. The drug dose was doubled after the completion of three cycles and the procedure was repeated until the final drug levels were achieved.All resistant cell lines were generated in our laboratory. The cells were seed in the concentration of 10 thousand cells/cmThe final concentrations used for selection were 1000 ng/ml Cis, 1100 ng/ml Pac, 100 ng/ml Dox, and 24 ng/ml Top. These concentrations were chosen based on the results of Dietel et al., 1997  and were2 atmosphere.All of the cell lines were maintained as monolayers in complete medium  at 37°C in a 5% COin vitro transcription (resulting in cRNA populations), biotin labelling, and cRNA fragmentation were performed using the same protocol.The RNA was isolated from the A2780, W1 cells and all of the resistant cells using TRIzol reagent, according to the manufacturer's protocol. The RNA was quantified using spectrophotometry by measuring the absorbance values at 260 nm and 280 nm, and the 260/280 nm ratio was used to estimate the level of protein contamination. The 260/280 nm ratios of the samples ranged from 1.8 to 2.0. The extent of RNA degradation was evaluated using an electrophoretic method employing a 1% denaturing agarose gel and by estimating the RIN factor using a Bioanalyzer 2100 system . RIN values ranged from 8.5 to 10, with an average value of 9.2. Additionally, each sample was diluted to a final working concentration of 100 ng/μl. All of the samples were prepared in triplicate. The cDNA for the microarray analysis was synthetized in two steps (separate synthesis for first and second strand) using an Affymetrix GeneChip® 3′ 'IVT Express Kit and 100 ng/μl of RNA, according to the Affymetrix GeneAtlas 3′ IVT Express Kit protocol. The next steps, which were The samples were loaded onto and hybridized to Affymetrix GeneChip Human Genome U219 microarrays, together with control cRNA and oligo B2. Hybridization was conducted at 45°C for 16 hours. using an AccuBlock™ Digital Dry Bath  hybridization oven Next, the microarrays were washed and stained according to manufacturer's protocol using an Affymetrix GeneAtlas™ Fluidics Station , and the chips were scanned using an Affymetrix GeneAtlas™ Imaging Station . The scans of the microarrays were saved as *.CEL files on hard disks.™  software, according to the manufacturer's standards. Secondary quality control studies were performed using Partek® Express™ Software . Gene-screening analysis of the QC-based results and statistical analysis (non-parametric Mann-Whitney test using α=0.05) were performed using the same software. As a result, a table showing the most significant fold changes in the levels of gene expression of the resistant cells relative to those of the parental cells was developed, after which it was imported into the Pathway Studio® Explore platform , in which pathway studies was performed. The genes with greatest fold changes in expression between the drug-resistant and parental cell lines were listed.Quality control (QC) studies were performed using Affymetrix GeneAtlashttp://www.r-project.org, version 2.14) Bioconductor (http://www.bioconductor.org) package.To visualize the effect of filtering the data, we applied the gene list to volcano plotting using a five-fold change in the expression level as the threshold (genes with expression upregulated more than 5-fold and downregulated -5-fold) Figure . threshoImmunohistochemical analysis was performed on transverse 5 μm formalin-fixed and paraffin embedded sections from human ovarian carcinoma placed on the SuperFrost/Plus microscope slides. We have investigated tissues from patients with ovarian serous adenocarcinoma and ovarian endometrioid adenocarcinoma. The analysis of EPHA7, IFI16, SPP1 and TGFBI expression was performed by use of the polymer-based immunohistochemical (IHC) technique  The prim2O2. The sections were incubated with appropriate primary antibodies overnight at 4°C and followed by incubation with EnVision Detection System Peroxidase/DAB, Rabbit/Mouse for 30 minutes . The sections were then counterstained with hematoxylin, dehydrated and mounted.The slides were dewaxed with xylene, and gradually hydrated. Activity of endogenous peroxidase was blocked by 30 minute exposure to 1% HHistological slides with protein expression were examined under an optical Olympus BH-2 microscope coupled to a digital camera. Color microscope images were recorded using LUCIA Image 5.0 computer software .The expression of given markers , was calculated taking into account the mean proportion of immunopositive cancer cells among all cancer cells counted in 10 light microscope fields each at magnification of 400x (at least 100 cancer cells per one microscopic field). Expression was evaluated using the semi-quantitative scale in which the score of 0 (negative) corresponded to no staining observed or less than 10% of cancer cells weakly positive; score 1 (weak) corresponded to 11% to 50% positive cancer cells; the score 2 (moderate) corresponded to 51% to 75% positive cancer cells; score 3 (strong) corresponded to up to 75% positive cancer cells.In summary, the alteration of the expression levels of many genes was observed in ovarian cancer cells in response to treatment with first- and second-line chemotherapeutic cytostatic drugs. Approximately half of these genes were not previously associated with any cancer including ovarian cancer and and not described in term of cytostatic-drug resistance. Changes in expression levels of these genes in three ovarian cancer cell lines that were resistant to the same cytostatic drug indicated that these changes might be specific markers of deleterious cellular responses to chemotherapy. Expression of selected genes was confirmed at protein level in ovarian cancer patients. Thus, up/downregulation of the expression of these genes pre- versus post-chemotherapy could be predictive in term of the response to treatment. For confirmation of these hypotheses, gene-expression studies should be conducted using an animal model of ovarian cancer and using samples of primary and metastatic tumours taken from ovarian cancer patients before and after chemotherapy is administered.


Establishment of continuous cell lines from human normal and tumor tissues is an extended and useful methodology for molecular characterization of cancer pathophysiology and drug development in research laboratories. The exchange of these cell lines between different labs is a common practice that can compromise assays reliability due to contamination with microorganism such as mycoplasma or cells from different flasks that compromise experiment reproducibility and reliability. Great proportions of cell lines are contaminated with mycoplasma and/or are replaced by cells derived for a different origin during processing or distribution process. The scientific community has underestimated this problem and thousand of research experiment has been done with cell lines that are incorrectly identified and wrong scientific conclusions have been published. Regular contamination and authentication tests are necessary in order to avoid negative consequences of widespread misidentified and contaminated cell lines. Cell banks generate, store and distribute cell lines for research, being mandatory a consistent and continuous quality program. Methods implementation for guaranteeing both, the absence of mycoplasma and authentication in the supplied cell lines, has been performed in the Andalusian Health System Biobank. Specifically, precise results were obtained using real time PCR detection for mycoplasma and 10 STRs identification by capillary electrophoresis for cell line authentication. Advantages and disadvantages of these protocols are discussed. The use of cultured cells that acquired the ability to proliferate indefinitely is an extended tool in research laboratories. Cell lines are used as in vitro models of health and disease by retaining many of the properties of the parental tissue or cell type, including disease-specific changes ]. The samples used were collected following informed consent of the donors and immediately anonymized. Local scientific and ethic committees approved the procedures performed in this work.mycoplasma detection. 100 µl supernatants were heated at 95 °C for 10 min and centrifuged at 1000 g for 5 s to discard cellular debris.Twenty-four supernatant samples from tumor cell lines generated by the Biobank were used to mycoplasma PCR Detection Kit  for detection of 19 mycoplasma species was used following the manufacturer instructions for mycoplasma detection in cell cultures. PCRs were performed using an Eppendorf AG thermocycler. Results were visualized using Agilent DNA 1000 Reagents  in a 2100 Bioanalyzer .LookOut mycoplasma qPCR Detection Kit  for detection of 66 mycoplasma species was used following manufacturer instructions for mycoplasma detection in cell cultures. PCR inhibition was discarded by an internal control from the kit (ROX labeled). PCRs were performed with specific Taqman probes (FAM labeled) using an ABI 7500 real time PCR thermocycler .LookOut For DNA isolation from blood samples, the paramagnetic beads based instrument Chemagic MSMI  was used. Briefly, Chemagic DNA Blood Kit special  was used for 3 ml of blood following manufacturer instructions. The corresponding Tris–HCl elution buffer available in the kits was used.For DNA isolation from tissues sections the paramagnetic beads based instrument Chemagic MSMI  was used. Chemagic DNA Blood Kit special  was used for tissue sections but with Proteinase K for tissue  and Lysis Buffer 1 for tissue . Between 10 and 18 20-µm sections for frozen tissues OCT were used (the exact number of sections varied with the area occupied by the tissue after hematoxylin staining). Tris–HCl elution buffer available in the kits was used.6 cells). QIAamp DNA Mini Kit  was used in a Qiacube robot following manufacturer instructions.Cell pellets were used for DNA isolation  following manufacturer instructions. Briefly, three washes were performed with Whatman® FTA® purification reagent followed by three washes with TE buffer. After TE buffer elimination, the FTA disc was dried during 1 h at room temperature. Dry FTA disc was used directly for multiplex PCR.The 1.2 mm Harris Uni-core punch  was used to obtain a FTA 1.2 mm disc that was introduced in a 0.2 ml PCR tube. DNA was purified from blood sample contained in the disc using the WhatmanMultiplex PCR for 5 STRs loci detection  was performed with primer sequences described by other author was performed using 10 ng of DNA isolated from cell lines, blood or tissue, or a pre-treated 1.2 mm FTA disc, and the GenePrint 10 System  according to the manufacturer instructions. STRs fragment detection was performed by capillary electrophoresis in a 3130 genetic analyzer  using the POP-7 matrix. Data analysis was carried out with the GemaMapper ID-X (v1.0.1) software .mycoplasma detection by conventional and real time PCR. By conventional PCR a 481 bp band was visualized for internal control detection in negative and positive samples and a specific 260 ± 8 bp band for mycoplasma positive samples . Corresponding results to the same donor (10 pairs of samples) were compared by both methods: 4 cell lines were compared with the original tissue, 3 cell lines were compared with frozen blood from the original donor, and 3 cell lines were compared with FTA punch. Coincident results were obtained for 5 STRs and 10 STRs loci detection methods except for pre-treated FTA discs, whose results were not precise through 5 STRs loci Multiplex PCR  have acquired at least one cell line from another laboratory, while 45% have never tested their cell lines for authenticity  of researchers that typically perform authentication testing in their laboratory  and 46% (115/250) of researchers have decided to perform mycoplasma and authentication analysis respectively, in the future    for DNA isolation was used to validate the Multiplex PCR methods through the distinct steps of cell line generation process. When we used 5 STRs and 10 STRs loci detection for checking cell lines authentication comparing to tissue and blood samples from the corresponding donors, good results were obtained of sensitivity and reliability. However reliable results were obtained for FTA punches tested with the 10 STRs Multiplex PCR method but not with the 5 STRs Multiplex PCR method. We hypothesize that it can be due to low DNA concentration in FTA punches, being probably the 10 STRs Multiplex PCR method more robust, sensitive and reliable for this kind of samples because of less DNA quantity required for STRs detection. Although technique complexity is higher, the fingerprint using 10 STRs loci provides an exact, sensitive, precise and objective result through capillary electrophoresis in an analyzer Table , which amycoplasma (Hancocks mycoplasma (Olarerin-George and Hogenesch Currently, prestigious scientific journals require evidence of cell lines authentication and absence of cross-contamination before data publication using immortalized cell lines (Lichter et al. Hancocks . HoweverHancocks , or clinHancocks . By othemycoplasma detection and authentication by validated methods of newly established or received cell lines prior to entering cell line collections is an essential issue. Literature and cell bank websites revision to find information about previous cross-contamination, and periodically testing of cell lines before cryopreservation and when thawed from liquid nitrogen is considered a good cell culture practice (Freshney mycoplasma in the supplied cell lines. This is the reason because highly recommended methods have been routinely introduced in the SSPA’s biobank for mycoplasma contamination and cell line authentication testing.In conclusion, Freshney . In case


Merkel cell polyomavirus (MCV) plays a causal role in ∼80% of Merkel cell carcinomas (MCC). MCV is clonally integrated into the MCC tumor genome, which results in persistent expression of large T (LT) and small T (sT) antigen oncoproteins encoded by the early locus. In MCV-positive MCC tumors, LT is truncated by premature stop codons or deletions that lead to loss of the C-terminal origin binding (OBD) and helicase domains important for replication. The N-terminal Rb binding domain remains intact. MCV-positive cell lines derived from MCC explants have been valuable tools to study the molecular mechanism of MCV-induced Merkel cell carcinogenesis. Although all cell lines have integrated MCV and express truncated LT antigens, the molecular sizes of the LT proteins differ between cell lines. The copy number of integrated viral genome also varies across cell lines, leading to significantly different levels of viral protein expression. Nevertheless, these cell lines share phenotypic similarities in cell morphology, growth characteristics, and neuroendocrine marker expression. Several low-passage MCV-positive MCC cell lines have been established since the identification of MCV. We describe a new MCV-positive MCV cell line, CVG-1, with features distinct from previously reported cell lines. CVG-1 tumor cells grow in more discohesive clusters in loose round cell suspension, and individual cells show dramatic size heterogeneity. It is the first cell line to encode an MCV sT polymorphism resulting in a unique leucine (L) to proline (P) substitution mutation at amino acid 144. CVG-1 possesses a LT truncation pattern near identical to that of MKL-1 cells differing by the last two C-terminal amino acids and also shows an LT protein expression level similar to MKL-1. Viral T antigen knockdown reveals that, like other MCV-positive MCC cell lines, CVG-1 requires T antigen expression for cell proliferation. Merkel cell carcinoma (MCC) is an aggressive skin cancer associated with immunosuppression and ultraviolet exposure . The incMerkel cell polyomavirus was discovered by digital transcriptome sequencing of MCC and was found to be clonally integrated in 70–80% of these tumors. While the viral integration sites in the tumor genome are random , MCC tumThese viral features in MCC tissues – clonal integration, LT helicase ablation, and T antigen expression - are also observed in the MCV-positive MCC cell lines. Since the first description of the MCV-positive MKL-1 cell line , 16 addiBoth MCC and small-cell lung carcinoma (SCLC) are neuroendocrine tumors, and MCC cell lines share cytological and histochemical similarities . AccordiDespite their similarities, MCV-positive MCC cell lines show variability in morphology, growth characteristics, and tumorigenicity. Mouse xenograft studies using four MCV-positive MCC cell lines have demonstrated significant differences in the latency required for grafted tumor cell lines to grow . In the Here, we describe the characteristics of a novel early passage MCV-positive MCC cell line, CVG-1, derived from a metastatic MCC explant and that exhibits LT truncation pattern and integrated viral copy number similar to that of the previously established MKL-1. While we found a unique leucine (L) to proline (P) missense mutation in the CVG-1-derived sT, this substitution mutation did not affect the transformation activity of CVG-1 sT in rodent fibroblasts. In fact, CVG-1 cell proliferation shows oncogenic addiction to viral T antigen expression similar to other MCV-positive MCC cell lines. Despite these similarities in virological features, a striking difference in tumor cell morphology was seen in CVG-1. Thus, establishment of CVG-1 not only extends the repertoire of MCV-positive cell lines but also serves as a tool to investigate host cell factors that contribute to the variations in growth morphology and tumorigenicity observed in other MCC cell lines.Metastatic MCC to the left cervical lymph nodes was surgically resected from a 70-year-old female with a previous history of MCC. After surgical resection, the tissue [Collaborative Human Tissue Network (CHTN)] was placed in the RPMI1640 medium. All specimens were tested under University of Pittsburgh Institutional Review Board-approved guidelines. Patient informed consent was obtained by the participating CHTN hospitals in accordance with the Declaration of Helsinki. Tumor tissue was minced to disaggregate tumor cells. To adapt cells to cell culture, cells were initially cultured in the RPMI medium containing 20% heat-inactivated fetal bovine serum (FBS), 0.01% penicillin/streptomycin, 0.01% fungizone, 0.01% insulin–transferrin–selenium, 50 μM bathocuproine disulfate, and 1 mM L-cysteine. After 9 days, cells were transferred into RPMI medium containing 10% FBS and non-essential amino acids . CVG-1 cells of passage <10 were cryopreserved in 10% DMSO in FBS. The experimental analyses in this study were carried out using cells at low passage (<40). Other MCC cell lines  were cultured in the RPMI medium supplemented with 10% FBS and NEAA.Formalin-fixed paraffin-embedded tumor tissue was stained according to a previously published protocol . BrieflyFigure 2C) or by CM8E6 .Cells were lysed in RIPA buffer  containing proteinase inhibitor cocktail (Roche). Lysates were sonicated to shear genomic DNA, and protein concentration was determined by the colorimetric DC-Protein Assay kit (Bio-Rad). After protein lysates were mixed with Laemmli buffer and heat-denatured, 30 and 50 μg of total protein were resolved in 12% SDS-polyacrylamide gel for LT and sT detection, respectively. MCV LT protein was detected by CM2B4 . MCV sT Genomic DNA was extracted from CVG-1 cells using standard phenol-chloroform extraction methods. The MCV DNA in CVG-1 cells was then amplified using polymerase chain reaction (PCR) primer sets (Contig1 to Contig13 primers) as described previously . PCR rea8 molecules).Subcellular fractionation was performed to isolate nuclei from MCC cells prior to genomic DNA extraction by Proteinase K/SDS buffer . After overnight incubation at 50°C, 100 ng/mL of RNaseA was added to the lysates and incubated for 1 h at 37°C. Extracted DNA was further purified by phenol-chloroform extraction and ethanol precipitation prior to quantitative PCR (qPCR). The obtained genomic DNA was dissolved in water and quantified by UV-Vis spectrophotometry. qPCR was performed to obtain absolute quantification using the aforementioned total T antigen primer set. In order to determine the absolute copy number, a dilution series of plasmid MCV-HF was used as a reference template DNA  per manufacturer’s instructions, treated with RNase-free DNaseI (NEB), and extracted again with Purezol reagent prior to cDNA synthesis. For cDNA synthesis, 500 ng of total RNA was subjected to first strand synthesis using the iScript cDNA synthesis kit (Bio-Rad). Subsequently, real-time PCR was performed using PowerUP SYBR Green Master mix to quantify total T antigen mRNA (sense: 5′-GCT CCT AAT TGT TAT GGC AAC AT-3′ and antisense: 5′-CAA CAT CCC TCT GAT GAA AGC-3′), sT mRNA (sense: 5′-TCC TTG GGA AGA ATA TGG AAC T-3′ and antisense: 5′-GCG AGA CAA CTT ACA GCT AAT AC-3′), and 18S rRNA (sense: 5′-GGA CAC GGA CAG GAT TGA CA-3′ and antisense: 5′-ACC CAC GGA ATC GAG AAA GA-3′). Real-time PCR reactions were triplicated for each sample and performed on the QuantiStudio 3 system (Applied Biosystems). The determined threshold cycle (Ct) values were used to calculate the mRNA abundance relative to CVG-1 using the delta-delta Ct method. The Ct values of 18S rRNA were used as reference.KpnI and EcoRI digestion, followed by displacing Cas9-2A-eGFP fragment with double strand DNA linker containing unique restriction sites using AgeI and NheI restriction sites. TLCV2 was a gift from Adam Karpf (Addgene #87360). Then, codon-optimized wild-type MCV sT and the amino acid substitution mutants including L114P, LSD, as well as L142A were inserted into pLenti TRE.MCS EF.Puro-2A-rTet by unique AgeI and SbfI restriction sites. For lentivirus production, 293FT cells (Invitrogen) were transfected with pLenti TRE MCV sT, psPax2, and pMD2.G using Lipofectamine 2000 (Invitrogen). The medium was changed at 12 h after transfection, and the lentivirus produced in the culture supernatant was harvested at 72 h after transfection.Rat-1 cell transformation assays were performed as described previously . For dox4) of the cells containing 0.3% noble agar (Sigma) was overlaid on the 2 mL of growth medium containing 0.6% agar. Cells were cultured for 2 weeks to observe colony formation in the soft agar. Colony numbers were counted under a microscope. The data is presented as mean ± standard deviation (SD) (N = 3).For the foci formation assay, Rat-1 cells were seeded in 6-well plate and infected with 150 μL of lentiviruses encoding multiple MCV sT variants. At day 3 post-infection, fresh growth medium with or without 0.5 μg/mL of doxycycline was added. Rat-1 cells were cultured for 11 days, and cell culture medium was refreshed every 3 days to maintain transgene expression. Cells were stained by 0.5% crystal violet to visualize transformation-associated foci. For soft agar colony formation assays, Rat-1 cells transduced with the aforementioned lentivirus constructs were treated with puromycin (3 μg/mL). After puromycin-resistant cells were selected, 1.0 mL (1.25 × 10DraI and EcoRV restriction sites. Oligonucleotides used to generate previously described shpanT (5′-CCG GAA GAG AGG CTC TCT GCA AGC TCT CGA GAG CTT GCA GAG AGC CTC TCT TTT TTG-3′ and 5′-AAT TCA AAA AAG AGA GGC TCT CTG CAA GCT CTC GAG AGC TTG CAG AGA GCC TCT CTT-3′) or control shRNA (5′-CCG GCC TAA GGT TAA GTC GCC CTC GCT CGA GCG AGG GCG ACT TAA CCT TAG GTT TTT G-3′ and 5′-AAT TCA AAA ACC TAA GGT TAA GTC GCC CTC GCT CGA GCG AGG GCG ACT TAA CCT TAG G-3′)  to produce pLenti e7SK-shpanT-puro and pLenti e7SK-shCtrl-puro. pLenti Dest-puro was constructed from pMuLE Lenti Dest-Neo (Addgene plasmid #62178) by replacing the neomycin phosphotransferase gene with the puromycin N-acetyltransferase gene containing SbfI and XhoI restriction sites. Lentivirus production was performed as described in transformation assays section. pENTR1A no ccdB (w48-1) and pLenti Dest Neo were a gift from Eric Campeau and Paul Kaufman and from Ian Frew, respectively.A modified version of the enhanced 7SK Pol III promoter (e7SK) was used as described previously . In ordeAG G-3′)  were ann4 cells were seeded in a 96-well plate (day 0). Cell proliferation was measured using WST-8 (Wako) at days 1, 3, 5, 7, 9, and 11. OD values were normalized by values from day 1. The WST-8 formazan product was measured at 440 nm with a reference filter at 600 nm using a Synergy 2TM Multi-Mode Microplate Reader (BioTek).For cell proliferation assays, CVG-1 cells were transduced with e7SK-shpanT-puro and pLenti e7SK-shCtrl-puro lentiviruses in the presence of 1 μg/mL polybrene for 2 days and selected for 4 days by puromycin treatment (1 μg/mL) as previously described . At day Figure 1A, Top panel). Immunohistochemistry using the MCV LT antibody (CM2B4) showed strong nuclear staining in both large and small tumor cells  confirming the presence of virus in each tumor cell.A small piece of the metastatic MCC tissue used to establish MCC cell line CVG-1, was formalin-fixed and embedded in paraffin for hematoxylin/eosin (H&E) staining and immunohistochemistry. MCC tumor cell generally exhibit cytological features of monomorphic small round cells with high nuclear to cytoplasmic ratio. However, H&E staining of CVG-1 showed a heterogeneous cell population accentuated by large tumor cells . We attempted to single-cell subclone large and small CVG-1 cells to demonstrate that both cells are LT-positive MCC but were unsuccessful. Nevertheless, since MCC tumor tissues are uniformly positive for LT protein, both large and small cells are likely to be MCV infected. CVG-1 cells grow in loose clusters in culture, whereas most MCC cells including MKL-1, MKL-2, and MS-1 grow in large, tightly cohesive aggregates . CVG-1 cells make contact with each other, but their contact involves only a small segment of the periphery of each cell.Similar to the morphologic features of the parental MCC tumor, the CVG-1 cell line also displayed tumor cell size variation, having a mixture of large and small round cells (Table 1). Although genome copy numbers are similar between CVG-1 and MKL-1, analyses using qRT-PCR demonstrated that total T antigen mRNA and sT mRNA expression levels approximately 2.5-fold higher in CVG-1 than in MKL-1 . MKL-2 expressed the lowest levels of T antigen mRNAs, consistent with previous results .To determine the sequence of the MCV genome in CVG-1 cells, we amplified the viral DNA from CVG-1 using 13 pairs of contiguous primer sets that span the entire region of the MCV genome . The expTable 1), there was no presence of multiple peaks/mixed signals, suggesting that all the copies of integrated MCV possess the identical truncation mutation. On the other hand, MKL-1 LT terminates at nucleotide position 1618 due to a 46-bp deletion that causes a frameshift. This frameshift mutation results in the addition of two unrelated amino acids prior to the termination codon. As their coding regions suggest, both CVG-1 and MKL-1 LTs are composed of 330 amino acids , and the only difference between these was found to be two amino acids at their C-terminal ends—Ser-Asn for CVG-1 and Lys-Leu for MKL-1. As observed in the MKL-1 LT, the premature truncation mutation found in the CVG-1 LT resulted in the complete deletion of the OBD and helicase domains without the affecting Rb-binding domain . The CVG-1 and MKL-1 cell lines are the first pair of cell lines that express the same length LT protein out of the nine MCV-positive MCC cell lines characterized to date. Next, we determined that the LT genetic truncation in CVG-1 has the same migration pattern as MKL-1 in SDS–PAGE . Consistent with the LT truncation pattern observed in the MKL-1 cell line, the CVG-1 LT migrated at 50 kDa. The mobility of CVG-1 LT, however, was marginally slower than that of MKL-1. Furthermore, LT levels in CVG-1 were slightly lower than those in MKL-1, whereas the T antigen mRNA levels in MKL-1 were higher than those in MKL-1 cells .Merkel cell carcinoma tissue-derived MCV harbors mutations that prematurely truncate the DNA helicase domain of LT . These tFigure 2C). These results suggest that LT and sT protein expression may be regulated at the post-transcriptional level.CVG-1 contains a unique missense mutation in the sT coding sequence (nucleotide position 536) that generates a Leu(L)-to-Pro(P) substitution at the amino acid position 114 of the sT protein. In the nine MCV-positive MCC cell lines in which the viral sequence has been defined, only one amino acid substitution has been found in three cell lines , all at position 20 of the sT protein (Ala (A) to Ser(S)). This position corresponds to a common exon1 of the T antigen gene, and thus, both LT and sT proteins are affected by the A20S substitution. Thus, L114P is a unique MCV sT amino acid substitution found in CVG-1. In contrast to LT, sT protein expression is relatively comparable across all the cell lines tested, irrespective of differences in mRNA expression (Table 1) tend to express higher LT protein than MKL-2 and MS-1 cell lines, which contain lower copy numbers of viral genome . We also confirmed protein expression of MCC marker CK20 by immunoblot. Consistent with previous results . In parallel with the foci formation assay, we performed a soft agar colony formation assay and observed anchorage-independent growth in cells expressing the L114P sT mutant . Colony size and numbers were comparable between L114P and the other transforming MCV sT variants tested, suggesting that the L114P mutation does not affect the transforming activity of MCV sT. Comparable sT protein expression in lentivirus-transduced cells was confirmed by immunoblotting .Merkel cell polyomavirus sT is the major MCV oncoprotein that causes loss of contact inhibition and confers anchorage-independent growth capabilities in immortalized rodent fibroblasts . This viFigure 3C). In addition, cell proliferation assays performed with WST-8 showed that T antigen knockdown by shpanT completely ablated the cell proliferation activity of CVG-1 cells . Thus, similar to most MCV-positive cell lines, tumorigenicity of CVG-1 also depends on the viral T antigen expression.While most MCV-positive MCC cell lines established to date require viral T antigen oncogene expression for proliferation, T antigen expression is dispensable only in one MCV-positive MCC cell line, LoKe . We examHere we report the establishment of a new, early passage MCV-positive MCC cell line CVG-1. As seen in other MCV-positive MCC cell lines MKL-1 and MCCLWhile the morphology of CVG-1 was unique among previously reported MCV-positive MCC cell lines, CVG-1 tumorigenicity is still dependent on MCV T antigen expression as in other MCV-positive cell lines. Out of seven cell lines tested for T antigen knockdown, only the LoKe cell line does not require T antigen expression for growth . Charactin vitro, but infection of Merkel cells has never been confirmed. Merkel cells are epidermal cells localized in the basal layer of the epidermis. While Merkel cells originate in the epidermis (in situ or Merkel cell hyperplasia (in situ, which is confined to the epidermis, may arise from Merkel cells (The cell of origin in MCC remains unknown. It has been thought to be the epidermal Merkel cells based on the similarities in ultrastructure and gene expression between normal Merkel cells and MCC . Howeverpidermis , cutaneopidermis . Recentlpidermis . Intereserplasia . These del cells .Since an animal model that mimics dermal MCC carcinogenesis has not been developed, MCC cell lines are useful tools to study the cellular origin of MCC. It has been shown that SV40 T antigen and human papilloma virus E6/E7 oncoproteins can reversibly transform primary human hepatocytes and human pancreatic duct epithelial cells without affecting normal diploid status . The MCVStudies using MCC cell lines have revealed critical oncogenic pathways regulated by sT and LT. A recent study demonstrated that MCV sT binds to L-Myc and the EP400 histone acetyltransferase complex to activate L-Myc-mediated gene expression in MCC cells critical for MCC cell proliferation . MCV LT We established a new, early passage MCV-positive MCC cell line CVG-1 from a patient with metastatic MCC. CVG-1 displays different morphologic features from other MCV-positive MCC cell lines, but nevertheless requires MCV T antigen for cell proliferation. While CVG-1 sT antigen contains a unique missense mutation, the mutant sT demonstrated similar transformation activity to prototypic sT in rodent cells. CVG-1 shows similarities to MKL-1 in viral copy numbers and LT truncation patterns. Further analyses of CVG-1 and MKL-1 may lead to the identification of critical host factors beyond the viral T antigen that contribute to the variations observed in MCC cell lines.CV, YC, and MS established the CVG-1 cell line. MS conceived and designed the experiments. YA performed the qPCR experiments. MS performed the sequencing experiments. YA, AH, and MS performed the immunoblot and cell transformation experiments. TT performed the immunohistochemistry. MS, YA, and YC analyzed the data. MS, YC, CV, and YA wrote the paper.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. The reviewer BA and handling Editor declared their shared affiliation.


Vaccinium virgatum Aiton; RB species), southern highbush blueberry , northern highbush blueberry , and wild blueberry  were compared. Of these, leaves of the RB species collected in December showed a significantly stronger inhibitory effect in both cell lines than the SB, NB, or WB species. These results suggest elevated biosynthesis of ATL-preventative bioactive compounds in the leaves of the RB species before the defoliation season.The inhibitory effects of blueberry leaves on the proliferation of adult T-cell leukemia (ATL) cell lines have previously been reported. A comparison of blueberry leaf extracts from different cultivars and seasonal variation were investigated regarding their effects on ATL cell line proliferation. The inhibitory effects of 80% ethanol leaf extracts from different blueberry cultivars collected from April to December in 2006 or 2008 were evaluated using two ATL cell lines. The bioactivities of leaf extracts of rabbit-eye blueberry ( Adult T-cell leukemia (ATL) occurs in a small population of individuals infected with human T-cell leukemia virus type I (HTLV-I). After infection with HTLV-I, 2% to 5% of carriers are likely to develop ATL after a long latency period of 30 to 50 years . AffecteVaccinium virgatum Aiton: RB species), southern highbush blueberry (V. spp.: SB species), northern highbush blueberry (V. corymbosum L.: NB species) and wild blueberry (V. bracteatum Thunb.: WB species), with several cultivars existing in each type. These species differ distinctly in cold resistance, height and fruit size [The leaves of blueberry, which belongs to the Ericaceae family, have been used as an anti-diabetic folk medicine in Europe and Canada ,6. Thereuit size . It is iuit size , ATL celAll blueberry cultivars used were cultivated in Miyazaki, Japan. All cultivars were taxonomically identified on the basis of morphological characteristics, and voucher specimens were deposited at the University of Miyazaki. The voucher numbers are provided in The fresh blueberry leaves were freeze-dried and powdered. The freeze-dried powders (approximately 10 mg) were extracted with 300 μL of 80% ethanol . The extracts were filtered using a centrifugal filter  (3000 rpm × 5 min). The filtrate was dried, dissolved in dimethyl sulfoxide, and diluted in RPMI 1640 medium prior to utilization in ATL cell proliferation assays. These vehicles did not affect the cytotoxicity of ATL cell lines from our preliminary experiments. The yields of the 80% ethanol extracts (% yield = (80% ethanol extracts (mg)/freeze dried powder (mg)) × 100) are presented in −1 penicillin G and 100 μg mL−1 streptomycin . Each cell line was subcultured twice a week, and cell numbers were adjusted to 1 × 105 cells mL−1 for the in vitro experiments.In this study, two ATL cell lines (ED and Su9T01), which are highly responsive to blueberry extracts based on previous results , were us5 cells mL−1, 90 μL per well) into a 96-well plate containing RPMI 1640 medium. After incubation at 37 °C for 24 h in an atmosphere containing 5% CO2, the blueberry extracts were added (10 μL per well) to the cells and incubated for an additional 72 h. Subsequently, the inhibition of cell proliferation was determined using a 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5--2H-tetrazolium monosodium salt (WST-8) assay kit . Viable cells convert the tetrazolium salt in WST-8 to the highly water-soluble formazan, which was monitored by measuring absorbance at 450 nm with a microplate reader . The sample results are presented as relative to control  × 100). Genistein  was used as a positive control [Each cell line was seeded . Statistical differences between the sample and genistein (positive control) groups were evaluated by analysis of variance (ANOVA) followed by Dunnett’s post-hoc test. Values with p < 0.05 were considered to be significant.Data are expressed as mean ± SD (−1) of three cultivars of the RB species , collected every month from April to December in 2006. Leaves were not collected during the fall season (from January to March). Among these cultivars, Homebell (50 μg mL−1), collected from October to December, showed significantly greater ED cell inhibition than genistein (50 μM), whereas from May to December, this cultivar (50 μg mL−1) showed significantly greater Su9T01 cell inhibition than genistein (50 μM) (−1), collected from July to December, showed significantly greater ED cell inhibition than genistein (50 μM), whereas from May to December, it showed significantly greater Su9T01 cell inhibition than genistein (50 μM)  A. Both M (50 μM) B,C. Thes−1) showed greater inhibitory activity than genistein (50 μM) (−1) showed a significantly greater inhibitory effect than genistein (50 μM) in ED cells. In Su9T01 cells, all cultivars of the RB species showed markedly greater inhibitory activity from June to December (50 μg mL−1) than genistein (50 μM) , five cultivars of the SB species , three cultivars of the NB species  and one cultivar of the WB species (Shashanbo). Unfortunately, due to poor growing conditions in 2008, Tifblue could not be collected. In ED cells, the RB species (except for Ethel and Homebell) from December (50 μg mL (50 μM) . In part (50 μM) .−1) than genistein (50 μM) in Su9T01 cells, which is in contrast to observations with ED cells. With respect to Su9T01 cell inhibition by the SB species, inhibitory effects were seen in the December samples of O’Neal and Reveille, the June to October samples of Sunshineblue, the October to December samples of Sharpblue, and the June to December samples of Flodablue. Regarding Su9T01 cell inhibition by the NB species, inhibitory effects were seen only in the April sample of Spartan, the April, October and December samples of Bluecrop, and the August to December samples of Berkeley. The WB species (Shashanbo) showed an inhibitory effect only in the April sample. Thus, samples from the SB, NB and WB species (50 μg mL−1) exhibited only a partially greater inhibitory effect than genistein (50 μM) in Su9T01 cells, unlike that observed with ED cells.Furthermore, Myers, Ethel, Bluebelle and Gardenblue showed a significantly greater inhibitory effect in April  [et al. reported that epigallocatechin-3-gallate inhibits proliferation of ATL, as well as HTLV-I-infected cells, by suppressing HTLV-I pX gene expression and inducing apoptotic cell death [Matsuo ns (PAs) . PAs, alns (PAs) . Li et all death . From thThe leaves of the RB species collected in December were the most effective inhibitors of proliferation in ATL cell lines. Thus, the best time to collect leaves of the RB species is before defoliation. This is the first study to extensively analyze differences in blueberry leaf extracts according to cultivar comparison and seasonal variation utilizing screening of ATL cell proliferation. Identification of the novel active compounds in blueberry leaves for use in the treatment and prevention of ATL will be undertaken in the future.


This study evaluates the tumorigenic and metastatic potential of a panel of three human FTC cell lines  with defined genetic mutations in two in vivo murine models: an orthotopic thyroid cancer model to study tumor progression and a tail vein injection model to study metastasis. All cell lines developed tumors in the orthotopic model, with take rates of 100%. Notably, WRO-derived tumors grew two to four times faster than tumors arising from the FTC-238 and TT2609-CO2 cell lines. These results mirrored those of a tail vein injection model for lung metastasis: one hundred percent of mice injected with WRO cells in the tail vein exhibited aggressive growth of bilateral lung metastases within 35 days. In contrast, tail vein injection of FTC-238 or TT2609-CO2 cells did not result in lung metastasis. Together, our work demonstrates that these human FTC cell lines display highly varied tumorigenic and metastatic potential in vivo with WRO being the most aggressive cell line in both orthotopic and lung metastasis models. This information will be valuable when selecting cell lines for preclinical drug testing.Follicular thyroid cancer (FTC) is the second most common type of thyroid cancers. In order to develop more effective personalized therapies, it is necessary to thoroughly evaluate patient-derived cell lines in  Follicular thyroid cancer (FTC) is the second most frequently diagnosed thyroid cancer after papillary thyroid cancer (PTC). About 10,000 cases of FTC are diagnosed annually in the United States, comprising about 10–15% of all thyroid cancer cases. The majority of FTC patients have excellent prognoses when their cancers are caught early. However, the relative 5-year survival rate drops dramatically to about 50% for patients with advanced metastatic cancer who does not respond to standard treatments. More research is needed to determine the cause of the disease; to study tumor initiation, progression, and drug resistance; and to eventually develop new and improved drugs for this group of patients.in vitro toxicology studies, as well as in vivo studies in immunodeficient murine models to study cancer progression and drug responses in the tumor microenvironment. Previously, we and other laboratories developed an orthotopic thyroid cancer model that faithfully recapitulates the clinical characteristics of advanced thyroid cancer  mice to initiate primary tumors. These cell lines were also injected into the lateral tail vein to evaluate their metastatic potential for lung colonization. Tumor progression and metastasis were evaluated by bioluminescent imaging weekly as well as postmortem histological analysis.In the current study, we characterize the tumorigenic and metastatic potential of a panel of three human FTC cell lines harboring various genetic mutations. The FTC-238 and TT2609-CO2 cell lines each has a  cancers . The WRO2:air mixture at 37°C.The WRO cell line, established from the metastases of a female FTC patient, and TT2609-CO2 cell line, established from a 57-year-old male patient with FTC, were kindly provided by Dr John Copland (Mayo Clinic) with permission from Dr Guy J F Juillard (UCLA) for the WRO cell line and Dr Albert A Geldof  for the TT2609-CO2 cell line. The FTC-238 cell line, established from a lung metastasis of a 42-year-old male FTC patient, was purchased from ATCC. All cell lines have been confirmed by short tandem repeat (STR) analysis. The WRO cell line was cultured in DMEM/F12 (1:1) with 10% fetal bovine serum (FBS). The TT2609-CO2 cell line was cultured in RPMI1640 with 10% FBS. The FTC-238 cell line was cultured in DMEM/ F12 (1:1) with 5% FBS. The cultures were maintained in a humidified chamber in a 5% CO6 cells with the RNeasy Kit (Qiagen) and treated with RNase-free DNase (Qiagen). Two micrograms of RNA were reverse transcribed into cDNA using the Thermoscript First-strand Synthesis System (Invitrogen). The oligonucleotide sequences of the primers for PAX8, TTF1 TSHR, TG, and TPO have been published elsewhere  to generate stable clones. A detailed description of the construction and transfection protocol has been published  and maintained under pathogen-free conditions at Saint Louis University Animal Facility. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Saint Louis University. All procedures were in accordance with institutional animal welfare guidelines, and all efforts were made to minimize suffering.Eight-week-old female NSG mice strains NOD.Cg5 cells were suspended in 10 μL Matrigel/RPMI or DMEM at a 1:1 dilution and injected into the right thyroid gland of NSG mice  and imaged with an IVIS Spectrum as described previously . However, unlike normal human thyroid, none of these cell lines had detectable levels of mRNA for the thyroid differentiation markers thyroglobulin (TG), thyroperoxidase (TPO), sodium-iodide symporter (NIS), or the receptor for thyroid stimulating hormone (TSHR)  were validated and obtained from the sources indicated in the Materials and methods section. The mutational characteristics of these human FTC cell lines, based on published reports , 8, 15, e (TSHR) , confirmluciferase-expressing cell lines suitable for in vitro and in vivo live imaging, WRO, FTC-238, and TT2609-CO2 cells were transfected with a pSIN-luc vector encoding a firefly luciferase gene, and three stable clones from each cell line were selected for further analysis. The levels of luciferase activity in serially diluted cell cultures were quantified in vitro using IVIS in complete media supplemented with luciferin. The results show that luciferase activity was proportional to the number of seeded cells  for all cell lines . Tumor initiation and growth were monitored weekly via bioluminescent live imaging using IVIS. Notably, WRO-derived tumors were more aggressive and invasive and grew two to four times faster than tumors arising from the FTC-238 and TT2609-CO2 cell lines. Tumor volume, assessed as total flux (photon counts per second), revealed that mice injected with WRO cells rapidly developed primary tumors within 7 days of injection. These mice survived a total of 14–18 days, whereas mice injected with FTC-238 cells survived a total of 27–29 days. In contrast, mice injected with TT2609-CO2 cells experienced significantly delayed onset of tumors and the tumors grew more slowly than those arising from WRO or FTC-238 cells. These mice survived a total of 57–70 days . In contrast, all mice injected with FTC-238 or TT2609-CO2 cells remained free of tumors 35 days after injection  on the growth of FTC-133 and FTC-238 cell lines .