Increased glycolysis is a hallmark of cancer metabolism, yet relatively little is known about this phenotype at premalignant stages of progression. Periodic ischemia occurs in the premalignant condition Barrett's esophagus (BE) due to tissue damage from chronic acid-bile reflux and may select for early adaptations to hypoxia, including upregulation of glycolysis.We compared rates of glycolysis and oxidative phosphorylation in four cell lines derived from patients with BE  in response to metabolic inhibitors and changes in glucose concentration. We report that cell lines derived from patients with more advanced genetically unstable BE have up to two-fold higher glycolysis compared to a cell line derived from a patient with early genetically stable BE; however, all cell lines preserve active mitochondria. In response to the glycolytic inhibitor 2-deoxyglucose, the most glycolytic cell lines (CP-C and CP-D) had the greatest suppression of extra-cellular acidification, but were able to compensate with upregulation of oxidative phosphorylation. In addition, these cell lines showed the lowest compensatory increases in glycolysis in response to mitochondrial uncoupling by 2,4-dinitrophenol. Finally, these cell lines also upregulated their oxidative phosphorylation in response to glucose via the Crabtree effect, and demonstrate a greater range of modulation of oxygen consumption.Our findings suggest that cells from premalignant Barrett's esophagus tissue may adapt to an ever-changing selective microenvironment through changes in energy metabolic pathways typically associated with cancer cells. Two well-known differences in energy metabolism have been shown to exist between normal and cancer cells: the Warburg and Crabtree effects. In 1927, Otto Warburg discovered that cancer cells produce twice as much ATP as normal cells through glycolysis, even in oxygenated tumors in vivo because periodic endoscopic surveillance is recommended in BE patients for early detection of cancer, allowing intermediate stages of intestinal metaplasia to be characterized and monitored at regular intervals in vivo have been derived from primary premalignant tissue at early and late stages of progression and provide an opportunity to study cellular metabolism of premalignant lesions in vitroBarrett's esophagus (BE) is a premalignant condition in which intestinal metaplasia replaces normal esophageal squamous epithelium and is associated with an increased risk of esophageal adenocarcinoma (EA) BE develops as an adaptation to the erosive environment of acid-reflux with periodic hypoxic events due to ulceration and ischemia Since metaplastic esophageal mucosa from individuals with BE have phenotypes suggesting selective adaptation to hypoxia, we hypothesized that later-stage BE cell lines display increased glycolysis and decreased oxidative phosphorylation, compared to early-stage cell lines. Also, since glucose levels would fluctuate with periodic tissue damage in BE we hypothesized that later-stage BE cell lines display the Crabtree effect. To test our hypotheses, we compared metabolic rates of glycolysis and oxidative phosphorylation in four cell lines derived from individuals with BE at different stages of progression and measured changes in response to metabolic inhibitors and varying glucose concentrations. If BE cell lines representing different stages of progression to cancer have distinct metabolic features, then it may be possible to develop novel metabolic biomarkers for EA risk or therapeutic targets for pre-malignant stages.Cell lines and control samples were derived from participants who were enrolled in the Seattle Barrett's Esophagus Study, which was approved by the Human Subjects Division of the University of Washington in 1983 and renewed annually thereafter with reciprocity from the Fred Hutchinson Cancer Research Center Institutional Review Board (FHCRC IRB) from 1993 to 2001. Since 2001, the study has been approved by the FHCRC IRB with reciprocity from the University of Washington Human Subjects Division.hTert-immortalized Barrett's esophageal cell lines CP-A, CP-B, CP-C and CP-D were previously derived from premalignant Barrett's esophagus tissue Since Barrett's epithelium is a metaplasia which is of different origin than normal esophageal squamous epithelium, there is no ‘normal’ tissue control; therefore CRL-4001/BJ-5ta , a genetically stable immortalized foreskin fibroblast cell line, was cultured as a control cell line in four parts of DMEM Dulbecco's Modified Eagle's Medium (Invitrogen), containing 4 mM L-glutamine and 4.5 g/L glucose, to one part of Medium 199 (Sigma), supplemented with 0.01 mg/ml hygromycin B  and 10% fetal bovine serum (Gemini Bioproducts). OE-33, an esophageal adenocarcinoma line  2, and passaged upon reaching 50–80% confluency, once to twice weekly. To minimize over-trypsinization, passaging was performed using 0.05% Trypsin-EDTA (Invitrogen).All cell lines were grown in a humidified 37°C incubator with 5% COSeveral esophageal adenocarcinoma cell lines in existing cell banks have been reported to be contaminated and/or misidentified The Seahorse XF24 flux analyzer  is capable of simultaneously measuring changes in pH and oxygen levels from cells seeded on 24-well plates. Extracellular acidification rates (ECAR), representative of glycolysis, and oxygen consumption rates (OCR), representative of oxidative phosphorylation, were measured on the Seahorse XF24. While carbon dioxide produced by cells also acidifies the culture medium, glycolysis accounts for approximately 80% of the ECAR, and therefore ECAR has been shown to be consistent with the rate of lactate production by cells Two days prior to seeding Seahorse plates, cell lines were passaged and seeded at cell densities that produce 50–70% confluency and consistent rates of proliferation. Prior to harvesting and re-seeding, the cell confluency was visually inspected.Since ECAR and OCR measurements with the Seahorse instrument are confined to the media beneath the fluorescent sensors, localized differences in cell density contribute to noise in the Seahorse assay. Thus, consistent cell seeding is critical to minimize measurement noise. Optimal cell densities were established for Seahorse plates by plating two seeding densities for each cell line and determining well-to-well variability in ECAR and OCR. Since ECAR and OCR were highly reproducible for each seeding density, to avoid the potential for over-growth, the lower of the two seeding densities was determined to be optimal. To plate cells for Seahorse measurement, cells were concentrated in 100 µl medium specific to the cell line (described in Tissue Culture methods) and gently seeded with a pipette tip positioned at a 45-degree angle into 24-well Seahorse culture plates (Seahorse Bioscience Inc.). Three hours later, cells had adhered to the wells and 150 µl of medium was added. After overnight growth, medium was replaced with buffer-free Seahorse assay medium: Dulbecco's Modified Eagle's Medium (Sigma) supplemented with 5 mM glucose, 2 mM glutamine (Invitrogen), 10 mg/L phenol red and 1.85 g/L NaCl . The plates were equilibrated for one hour in a 37°C room-air incubator and sensors were calibrated in calibration solution (Seahorse Bioscience Inc). The plates were then evaluated on the Seahorse XF24. Five consecutive measurements were obtained on each well; however since the first OCR measurement consistently varied by more than two standard deviations from the subsequent measurements, it was discarded. Outlier data from wells that produced two-fold or greater increases in ECAR or OCR standard deviation when pooled were excluded from the analysis, since noisy sensors or poor signal from uneven seeding were suspected. Data from three to four wells per experiment were combined across at least six experiments for each cell line.http://www.stata.com).Preliminary analysis was performed using the Seahorse XF24 software (Version 1.7.0.74). Statistical analyses of differences in ECAR and OCR between cell lines were performed using ANOVA and Tukey-Kramer test on STATA software , a glucose analogue that competitively inhibits hexokinase activity in the first enzymatic step of glycolysis. Changes in ECAR and OCR were measured on the Seahorse XF24 in response to addition of 2-DG in consecutive injections that produced final concentrations of 1, 10, 25 and 50 mM. Five consecutive measurements were performed per well prior to and after each addition. Differences between cell line changes in ECAR and OCR at 50 mM 2-DG were analyzed with ANOVA and Tukey-Kramer tests using STATA software.2,4-dinitrophenol , a well-established protonophore and mitochondrial uncoupling agent, causes depolarization of the inner mitochondrial membrane, resulting in a dramatic arrest of ATP generation. Cell line changes in ECAR and OCR were measured on the Seahorse XF24, in response to addition of 2,4-DNP in consecutive injections that produced final concentrations of 2, 20, 50 and 200 µM. Five consecutive measurements were performed per well, prior to and after each addition. In most cell lines, 200 µM 2,4-DNP produced a sudden collapse of OCR below baseline levels, indicating toxicity, thus only data from 2 to 50 µM 2,4-DNP were used for further analysis. Differences between cell line changes in ECAR and OCR at 50 µM 2,4-DNP were analyzed with ANOVA and Tukey-Kramer tests using STATA software.Oligomycin, an ATP synthase inhibitor, was used to measure the amount of OCR which is dependent on ATP synthesis in each cell line. Cell line changes in ECAR and OCR were measured on the Seahorse XF24, in response to addition of 3.2 µM oligomycin. Five consecutive measurements were performed per well, prior to and after addition of the inhibitor. By subtracting the residual OCR following inhibitor addition from the total OCR prior to treatment, the OCR dependent on oxidative phosphorylation was obtained.Cell lines were cultured in Seahorse plates as described above, except glucose-free buffer-free assay medium was substituted for the standard Seahorse assay medium. Following room air and buffer-free media equilibration and sensor calibration, the plates were run on the Seahorse XF24 instrument. Cell line changes in ECAR and OCR were measured in response to addition of glucose in consecutive injections that produced final concentrations of 0.25, 0.5 and 5 mM. CCCP  was then added at 0.5 µM final concentration to verify that mitochondria remained functional despite a drop in OCR following glucose addition. Although CCCP has a lower dynamic range than 2,4-DNP, its purpose in this experiment was to fully uncouple the mitochondria and not titrate. Five consecutive measurements were obtained prior to and following each addition. Data were evaluated for inter-replicate variability as above and pooled from four experiments. Differences between cell line changes in ECAR and OCR at 5 mM glucose were analyzed with ANOVA and Tukey-Kramer tests using STATA software.http://rsbweb.nih.gov/ij/index.html) with a software macro that identified and counted nuclei. In each well, cells were imaged in four separate fields of view, each located in a distinct quadrant and visually verified to contain no staining artifacts. The mean from the four fields was extrapolated to the area of the well to estimate total number of cells. ECAR and OCR values were normalized using these cell counts within the Seahorse XF24 software (Version 1.7.0.74).Following each experiment, Seahorse assay media was replaced with 50 µl freezing media  with 10% DMSO (Sigma), 5% heat deactivated fetal calf serum, 5 mM Hepes Buffer) and stored at −25°C. On the week of cell counting, plates were thawed, washed with PBS at room temperature, fixed with 70% ethanol for 10 minutes at 4°C, washed again with PBS and stained with 5 µg/ml DAPI. The plates were stored at 4°C in the dark until data collection. Image data were acquired using a Nikon-Ti microscopy platform equipped with a Sutter Instruments Lambda LS 300 W xenon arc lamp, 360/BP40 excitation and 460/BP50 emission filters, and automated stage. Image cytometry analysis was performed using ImageJ  using Illumina Omni-quad 1 M SNP arrays. DNA was isolated from an early passage of each cell line as well as from constitutive normal tissue (blood or gastric tissue) from the individual from which each cell line originated. Samples were processed and hybridized according to manufacturer's protocol. Data were initially processed using Illumina Bead Studio, and regions of gain, loss and copy neutral LOH were determined using Partek Genomics Suite v6.5.EPC-2, CRL-4001 or OE-33 were excluded from nuclear genome characterization since they did not have patient-matched normal controls.Cell line mitochondrial genomes were assessed using MitoChip v2.0 oligonucleotide arrays from Affymetrix. Sequences were compared to matched constitutive normal samples to ensure the alterations detected were somatic changes. Since EPC-2, CRL-4001 or OE-33 did not have patient-matched normal controls, they were excluded from mitochondrial genome characterization. For cell lines, DNA was extracted by standard protocol on Qiagen columns . For constitutive DNA, DNA was extracted from gastric biopsies using Qiagen columns or Gentra DNA isolation kit . mtDNA was amplified using two sets of PCR primers and samples were quantitated, fragmented and labeled according to the MitoChip v2.0 protocol.MitoChip data were then analyzed using Affymetrix GCOS v1.3d and GSEQ v4.1 software and the putative mutations were verified on both forward and reverse strands. Cell line alterations that were detected were compared with matched constitutive DNA. Further characterization of the location of mutations, affected mitochondrial genes, and mitochondrial haplogroup for each cell line were determined using the Mitomaster mtDNA sequence analysis program All cell lines were grown to 50–70% confluency and harvested with 0.05% trypsin. Cells were centrifuged at 100 g for six minutes and resuspended in KSFM media (Invitrogen) to a concentration of 4 million cells/ml.To assess mitochondrial mass, cells were then diluted 1∶2 and stained for 30 minutes at 37°C in a staining solution containing final concentrations of 10 nM mitotracker green, 10 µM Hoechst 33342 and 5 µM Sytox Orange . Samples were measured by flow cytometry on the Beckton-Dickinson LSR-2 instrument. Dead cells were gated out using Sytox Orange, and were less than 0.1% for each cell line. Analysis was performed using FlowJo software  on whole-cell and G1-phase fractions that were gated using the Hoechst 33342 cell cycle profile.To determine if late-stage BE cell lines demonstrate increased glycolysis and/or decreased oxidative phosphorylation compared to an early-stage BE cell line, extracellular acidification rates (ECAR) and oxygen consumption rates (OCR) were measured using the Seahorse XF24 analyzer in an early-stage BE cell line (CP-A), three late-stage BE cell lines , and a fibroblast control cell line (CRL-4001). Difficulties associated with cell adhesion prevented normalization of the EA cell line OE-33 and comparison of absolute OCR and ECAR to the BE cell lines, however relative results were still reported .−7; −7 and p<0.001 respectively; ECAR in CP-D was two-fold higher than in CP-A p<10−7; . ECAR inTo confirm that OCR is dependent on ATP synthesis in each cell line, ECAR and OCR changes were also measured in response to treatment with the ATPase inhibitor oligomycin. All cell lines demonstrated a 57–73% decrease in OCR, which corresponds to their OCR that is dependent on ATP synthesis by oxidative phosphorylation . In respTo test the effects of glycolytic inhibition on the BE cell lines, ECAR and OCR changes were measured in response to 2-deoxyglucose (2-DG), a competitive inhibitor of the glycolytic pathway. If a cell line has elevated glycolysis (such as CP-C and CP-D), a greater decrease in ECAR would be expected in response to 2-DG compared to cells that are less glycolytic. In cell lines that have functional mitochondria, OCR would also increase to compensate for the decrease in glycolytic ATP production. If OCR does not increase in response to glycolytic inhibition, then the cells would have adequate ATP production by oxidative phosphorylation alone, have dysfunctional mitochondria or are unable to oxidize alternative substrates (eg. glutamine or fatty acids). If OCR instead decreases in response to glycolytic inhibition, then the rate of oxidative phosphorylation in that cell line would likely be limited by the rate of synthesis of pyruvate or other mitochondrial substrates.−4 and p<10−7 respectively; In response to 2-DG, ECAR decreased more in CP-C and CP-D than in CP-A  dissipate mitochondrial potential and arrest ATP generation. In cases where mitochondrial potential exceeds the reversal potential of the adenine nucleotide translocase (ANT), mitochondrial ATPase would consume ATP to pump protons across the inner mitochondrial membrane to restore potential −7 respectively; −7 and p<0.05 respectively; In response to 2,4-DNP, CP-C and CP-D had a lower increase in ECAR than CP-A  and lowest in the cell line derived from a patient with relatively more stable genome (CP-A), suggesting that a glycolytic phenotype was selected during the process of neoplastic progression. The BE cell line with the highest genomic instability, CP-D, was more glycolytic than the other BE cell lines, which was consistent with it having the greatest ECAR decrease following 2-DG addition and lowest ECAR increase following 2,4-DNP addition. However, mitochondrial function was conserved during CP-D's ‘glycolytic switch’ since it was able to compensate for glycolytic inhibition by upregulating oxidative phosphorylation. Although CP-C was more glycolytic than CP-A, it displayed a high level of OCR, which did not increase in response to glycolytic inhibition. This greater reliance on oxidative phosphorylation made CP-C more insensitive to glycolytic inhibition than CP-D. Similar to CP-D however, ECAR in CP-C was more insensitive to uncoupling by 2,4-DNP than CP-A, which is consistent with the higher baseline ECAR in CP-C. Insensitivity of glycolysis rates in CP-D to mitochondrial inhibition was also confirmed using the ATPase inhibitor oligomycin . All celOCR in the BE cell lines did not correlate with ECAR, indicating that the changes in the two metabolic pathways evolved independently. The lack of OCR increase in CP-A, CP-B and CP-C cell lines in response to glycolytic inhibition suggests that ATP/ADP levels did not fall below a threshold that would stimulate mitochondrial activity, suggesting that oxidative phosphorylation supplied the majority of energy in these cell lines. Likewise, in response to 2,4-DNP-mediated uncoupling, OCR increases were not significantly different in CP-A and CP-C, and marginally higher in CP-D, suggesting that their mitochondria maintained a similar level of mitochondrial reserve capacity. This was also confirmed in an independent experiment in which oligomycin-treated BE cells had similar percentage OCR decreases, which corresponds to a similar contribution of ATP-generating coupled-respiration to total cellular OCR . CP-B, tFinally, CP-C and CP-D cell lines demonstrated a greater change in OCR via Crabtree effect than the less glycolytic CP-A and CP-B cell lines, suggesting adaptation towards a more cancer-like phenotype. Increased Crabtree effect in BE cells predicts a greater survival advantage to adapt to conditions of glucose and oxygen fluctuation such as gastric reflux-induced ulceration or ischemia.Difficulties associated with OE-33 cell adhesion prevented normalization of data collected for this EA cell line for direct comparison to the BE cell lines. However, based on normalization estimated from a limited data set, OE-33 had the highest levels of ECAR out of the cell lines tested and had percent ECAR that decreased as much as CP-D following 2-DG addition, indicating that it is a significantly glycolytic cell line . OE-33 aGiven our findings, we propose a model in which BE begins with a metabolism which is largely dependent on oxidative phosphorylation, as seen in CP-A, but then progresses through a metabolic phenotype which is intermediate between normal and cancer . This inSeveral mechanisms are likely to contribute to the metabolic changes in the BE cell lines. Both copies of TP53 are mutated and/or altered in CP-B, CP-C, and CP-D , which mSince endogenous reactive oxygen species (ROS) are produced by mitochondria, the mitochondrial genome is particularly susceptible to ROS-mediated damage. Although CP-A had the fewest nuclear genome alterations , it dispAlthough the Warburg and Crabtree effects have been commonly associated with cancer progression, higher glycolysis and Crabtree effect have also been observed in actively proliferating non-cancerous cells, such as stem cells and lymphocytes in vivo are from BE tissue quantitative fluorescent imaging which displays elevated NAD(P)H levels representative of glycolytic tissue Given the preliminary nature of our findings, it is important to determine if the metabolic changes found in BE cell lines are also found in biopsies from patients. Although tissue culture is clearly a model system, all of the metabolic measurements of ECAR and OCR were done at confluency, a crude approximation of the structure of cells in tissue. Also, the BE cell lines in this study were immortalized using hTERT, avoiding potential metabolic effects of other immortalization methods that affect p53, Rb, and Myc activity such as SV40 large T antigen. However, since the BE and EA cell lines used in this study were derived from different patients, further analysis of longitudinally collected samples within the same patient would more directly address these questions. The most promising support for our findings Figure S1Barrett's esophagus cell lines CP-B, CP-C and CP-D display higher genome instability than CP-A. Genome copy number alterations, relative to normal patient-derived diploid matched control , were plotted for each of the cell lines. CP-B, CP-C and CP-D display a large number of chromosomal alterations, compared to CP-A. Black dots represent moving averages of copy number; Gray dots represent individual locus copy number.(TIF)Click here for additional data file.Figure S2Barrett's esophagus cell lines are not significantly different in mitochondrial mass. Representative experiments are shown with (a) profiles of relative mitotracker green intensity  for each of the cell lines, gated on live-fraction by Sytox Orange and G1-fraction by Hoechst 33342 staining; and (b) comparisons of mean mitotracker ratios from two repeat experiments. Error bars represent standard deviation between experiments (N = 2). Comparable results are obtained when non-G1 fractions are included in the analysis.(TIF)Click here for additional data file.Table S1BE cell lines display genomic alterations that confer risk of progression  or are genetically unstable . Deletions in genes that confer risk of progression to EA are reported. Mutations in p16 and TP53 were determined by Palanca-Wessels et al * The patient from whom CP-B was derived also had EA detected in the same endoscopy.(DOCX)Click here for additional data file.Table S2Analysis of copy number alterations of genes involved glycolysis, oxidative phosphorylation and hypoxia regulation in BE cell lines. Gene symbol is the human genome Gene Symbol for the gene. Copy number gains are marked as ‘+’ and single copy losses as ‘−’. No double copy losses were detected in the genes investigated. *Note that prolyl 4-hydroxylases are inhibitors of HIF-1 mediated hypoxic resistance.(DOCX)Click here for additional data file.Table S3Effects of 2-DG treatment on ECAR and OCR in cell lines. The mean changes in ECAR and OCR after addition of 50 mM 2-DG compared to untreated baseline measured by Seahorse XF24 (N = 2–4). Abbreviations: SD = standard−deviation of means; p-value (Tukey-Kramer test) of statistically significant differences from CP-A are shown.(DOCX)Click here for additional data file.Table S4Effects of 2,4-DNP and Oligomycin on ECAR and OCR in cell lines. The mean changes in ECAR and OCR after addition of 50 µM 2,4-DNP compared to untreated baseline measured by Seahorse XF24 (N = 2–4). Abbreviations: SD = standard−deviation of means; p-value (Tukey-Kramer test) of statistically significant differences from CP-A are shown.(DOCX)Click here for additional data file.Table S5Effects of glucose on changes in ECAR and OCR in cell lines via the Crabtree effect. The mean changes in ECAR and OCR after addition of 5 mM glucose compared to glucose-free baseline measured by Seahorse XF24 (N = 2–4). Abbreviations: SD = standard−deviation of means; p-value (Tukey-Kramer test) of statistically significant differences from CP-A are shown.(DOCX)Click here for additional data file.Table S6Analysis of copy number alterations of genes involved glycolysis, oxidative phosphorylation and hypoxia regulation in BE cell lines. Gene symbol is the human genome Gene Symbol for the gene. Copy number gains are marked as ‘+’ and single copy losses as ‘−’. No double copy losses were detected in the genes investigated. *Note that prolyl 4-hydroxylases are inhibitors of HIF-1 mediated hypoxic resistance.(DOCX)Click here for additional data file.Table S7Summary of copy number alterations in nuclear-encoded mitochondrial genes. 1023 mitochondrial genes were characterized for deletions, gains of copy number, and copy neutral loss of heterozygosity (LOH).(DOCX)Click here for additional data file.Table S8Summary of mitochondrial mutations in BE derived cell lines. Nucleotides for mutation locations and haplogroup designation based upon data from Mitomaster mitochondrial mutation database and analysis tool * indicates mutations that share their location with a known human mitochondrial polymorphism.(DOCX)Click here for additional data file.Text S1BE and control cell line background and characterization. This file contains the background and characterization of four BE cell lines derived from BE segments, three genetically-stable control cell lines, and cell line derived from an adenocarcinoma.(DOCX)Click here for additional data file.Text S2Nuclear and mitochondrial genome characterization. This file contains additional details from the nuclear and mitochondrial genome analyses performed.(DOCX)Click here for additional data file.


Transgenic cattle carrying multiple genomic modifications have been produced by serial rounds of somatic cell chromatin transfer (cloning) of sequentially genetically targeted somatic cells. However, cloning efficiency tends to decline with the increase of rounds of cloning. It is possible that multiple rounds of cloning compromise the genome integrity or/and introduce epigenetic errors in the resulting cell lines, rendering a decline in cloning. To test these possibilities, we performed 9 high density array Comparative Genomic Hybridization (CGH) experiments to test the genome integrity in 3 independent bovine transgenic cell lineages generated from genetic modification and cloning. Our plan included the control hybridizations (self to self) of the 3 founder cell lines and 6 comparative hybridizations between these founders and their derived cell lines with either high or low cloning efficiencies.We detected similar amounts of differences between the control hybridizations  and the comparative analyses of both "high" and "low" cell lines (ranging from 7 to 57 with a mean of ~20). Almost 75% of the large differences (>10 kb) and about 45% of all differences shared the same type (loss or gain) and were located in nearby genomic regions across hybridizations. Therefore, it is likely that they were not true differences but caused by systematic factors associated with local genomic features (e.g. GC contents).Our findings reveal that large copy number variations are less likely to arise during genetic targeting and serial rounds of cloning, fortifying the notion that epigenetic errors introduced from serial cloning may be responsible for the cloning efficiency decline. As embryonic stem cells are not available in the bovine species, somatic cells have been used for genetic modifications, and transgenic cattle have been produced from such genetically modified somatic cells by animal cloning. However, because primary somatic cells have limited life span and inevitably become senescent following DNA transfection and selection in cell culture, it is impossible to perform any further genetic modifications in these cells. Because of such, transgenic cattle with a desired genotype that requires more than one genetic targeting event, such as homozygous deletion of the two alleles of a gene, cannot be produced. To overcome such limitations, a novel sequential genetic modification strategy in bovine somatic cells, for producing extensively genetically modified cattle, has been developed . This prTo investigate whether the declines of cloning efficiency in the cloned bovine transgenic cell lines are due to large genomic deletions or insertions, 9 high density array Comparative Genomic Hybridization (CGH) experiments were performed to test the genome integrity in 3 independent bovine transgenic cell lineages. Array CGH allows the entire genome to be assayed for the gain or loss of material in a single experiment by measuring the relative hybridization intensity between fluorescently labeled test and reference DNA samples. It has been widely used in the detection of copy number variations (CNVs). One objective of this study is to develop array CGH into a systematic test for the genomic integrity of donor cells after each round of genetic modification before they are used as donors for producing transgenic animals.http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE26132.We selected 3 independent cell lineages from our transgenic bovine cell line collection. Each lineage includes the founder and two derived cell lines, which demonstrated dramatic differences in cloning efficiency Figure . The cloWe detected 8, 13 and 39 differences in 3 control hybridizations. Similar amounts of differences (ranging from 7 to 57 with a mean of ~20) were detected in comparative analyses of both "high" and "low" derived cell lines  during animal cloning, supporting our recent findings that epigenetic errors introduced by multiple rounds of cloning and/or genetic targeting are the possible underlying causes for the cloning efficiency decline ,9. HowevYK and ZW are employees of Hematech, Inc., a subsidiary of Kyowa Hakko Kirin Company, Ltd. The authors declare that they have no competing interests.in silico prediction and computational analyses. GEL, YK and ZW wrote the paper.GEL and ZW conceived and designed the experiments. JMR provided reagents. GEL and YH performed All authors have read and approved the final manuscript.


Pheochromocytomas (PCCs) are slow-growing neuroendocrine tumors arising from adrenal chromaffin cells. Tumors arising from extra-adrenal chromaffin cells are called paragangliomas. Metastases can occur up to approximately 60% or even more in specific subgroups of patients. There are still no well-established and clinically accepted “metastatic” markers available to determine whether a primary tumor is or will become malignant. Surgical resection is the most common treatment for non-metastatic PCCs, but no standard treatment/regimen is available for metastatic PCC. To investigate what kind of therapies are suitable for the treatment of metastatic PCC, animal models or cell lines are very useful. Over the last two decades, various mouse and rat models have been created presenting with PCC, which include models presenting tumors that are to a certain degree biochemically and/or molecularly similar to human PCC, and develop metastases. To be able to investigate which chemotherapeutic options could be useful for the treatment of metastatic PCC, cell lines such as mouse pheochromocytoma (MPC) and mouse tumor tissue (MTT) cells have been recently introduced and they both showed metastatic behavior. It appears these MPC and MTT cells are biochemically and molecularly similar to some human PCCs, are easily visualized by different imaging techniques, and respond to different therapies. These studies also indicate that some mouse models and both mouse PCC cell lines are suitable for testing new therapies for metastatic PCC. SDHB) -dopamine ([18 F]-DA) [123I]-MIBG uses similar mechanisms to enter these cells, but importantly, its affinity to NET is lower compare with [18 F]-DA. Uptake in these tumors depends on both NET and VMAT and subsequently storage in vesicles. Therefore, if PCC metastases do not express NET, other (less specific) radiotracers can be used for detection and localization of tumors, including [18 F]-DOPA. [18 F]-DOPA is an analog of l-3,4-dihydroxyphenylalanine (l-DOPA) [18 F]-DA and [18 F]-DOPA PET performed equally well for the detection of ovarian metastatic tumors, [18 F]-DOPA PET showed superiority to [18 F]-DA PET in the detection of hepatic, lung, and subcutaneous tumors. In particular, subcutaneous tumors were detected only with [18 F]-DOPA PET. Comparisons of the in vitro uptake of both radiotracers by MPC cells confirmed the importance of the NET for uptake of [18 F]-DA and also confirmed that VMAT2, and to a lesser extent VMAT1, is important for its retention in catecholamine storage vesicles. The differences in uptake of these two radiotracers may be relevant to recent clinical findings of differences in functional imaging characteristics of various PCC, depending on their underlying mutations and nature [18 F]-DA is superior to [18 F]-DOPA for localization of metastases , whereas in other patients with so-called head-and-neck paragangliomas, [18 F]-DOPA is superior to [18 F]-DA -DA) , 72 ente8 F]-DA) , 74 and (l-DOPA) , a precu(l-DOPA)  demonstrd nature . For exa18 F]-DA .Recently, another model of metastatic PCC has been developed, following the approach revealed from gene expression differences between subcutaneous and liver metastases . Liver lP = 0.0027, and KRT8, P = 0.0003) that were significantly downregulated in MTT cells compared with MPC confirming the microarray analysis -DA functional imaging and identifying of NET expression in metastases in the metastatic mouse model allowed utilizing this animal model in experimental treatment through the modification of NET expression in PCC. One of the most effective treatments for malignant PCC includes [131I]-MIBG [131I]-MIBG [131I]-MIBG concentrations within the tumor cells. Thus, an increase of NET and of the number of storage granules might improve the response rate of such treatment. Liver tumors in MPC mouse model revealed a wide range of NET expression as well as [123I]-MIBG/[18 F]-DA uptake [123I]-MIBG/[18 F]-DA. In general, those positive on [123I]-MIBG scintigraphy are good candidates for [131I]-MIBG treatment, but there is a need to treat also those patients with negative [123I]-MIBG scintigraphy. After series of tests, a pretreatment with a single dose of romidepsin, 2.5 mg/kg, was used to evaluate its effect on isotope accumulation in the tumors. In conclusion, treatment with the HDAC inhibitors romidepsin and trichostatin A increased [123I]-MIBG and [18 F]-DA uptake in MPC cells in vitro and in vivo in liver metastatic lesions, through the upregulation of the cell membrane NET [131I]-MIBG in patients with advanced malignant PCC, paraganglioma, and other related tumors such as neuroblastoma.The advantage of positive results from [1I]-MIBG –90 that 1I]-MIBG , 89, 91.1I]-MIBG . This diA uptake . Testingrane NET . These dThe failure of cytotoxic cancer regimens to cure the most drug-resistant, well-differentiated solid tumors has been attributed to the heterogeneity of cell types that differ in their capacity to grow, differentiate, and metastasize. Data also supports that MPC cells and PCC metastasis are in different cell cycle phases, and thus any experimental treatment approach would have only short-term responses. A new approach of investigating the effect of LB1, a small molecule inhibitor of serine/threonine protein phosphatase 2A (PP2A), was proposed and presented by Lu et al. , in a nePten knock-out mice, but this has not been successful yet. As an alternative, the MPC and MTT cell lines could be used, which have been created from radiated Nf knock-out mouse PCC. Both the spontaneous metastatic PCC mouse models and mice injected with MPC and MTT cell lines might be useful for the investigation of currently available targeted cancer therapies.PCC of several animal models have been shown to resemble human PCC. Most mouse and rat models presented with benign PCC, but two studies reported lung metastases. Because no human PCC cell line exists, other cell lines would be very relevant to determine what chemotherapeutics could be used for the treatment of metastatic PCC. It would be beneficial to create cell lines of the “spontaneously” metastasizing PCC of


Cm = 1.9 µF/cm2. In contrast, cell lines carrying mutations in both p53 and PTEN (U373-MG and SNB19) showed the highest Cm values of 3.7–4.0 µF/cm2, which corroborate well with their heavily villated cell surface revealed by SEM. Since PTEN and p53 are well-known inhibitors of mTOR, the increased membrane area/folding in mutant GBM lines may be related to the enhanced protein and lipid synthesis due to a deregulation of the mTOR-dependent downstream signaling pathway. Given that membrane folds and extensions are implicated in tumor cell motility and metastasis, the dielectric approach presented here provides a rapid and simple tool for screening the biophysical cell properties in studies on targeting chemo- or radiotherapeutically the migration and invasion of GBM and other tumor types.Glioblastoma multiforme (GBM) is characterized by rapid growth, invasion and resistance to chemo−/radiotherapy. The complex cell surface morphology with abundant membrane folds, microvilli, filopodia and other membrane extensions is believed to contribute to the highly invasive behavior and therapy resistance of GBM cells. The present study addresses the mechanisms leading to the excessive cell membrane area in five GBM lines differing in mutational status for PTEN and p53. In addition to scanning electron microscopy (SEM), the membrane area and folding were quantified by dielectric measurements of membrane capacitance using the single-cell electrorotation (ROT) technique. The osmotic stability and volume regulation of GBM cells were analyzed by video microscopy. The expression of PTEN, p53, mTOR and several other marker proteins involved in cell growth and membrane synthesis were examined by Western blotting. The combined SEM, ROT and osmotic data provided independent lines of evidence for a large variability in membrane area and folding among tested GBM lines. Thus, DK-MG cells (wild type p53 and wild type PTEN) exhibited the lowest degree of membrane folding, probed by the area-specific capacitance  Glioblastoma multiforme (GBM) is the most common and aggressive human brain cancer, accounting for about 15% of all intracranial tumors Numerous light and electron microscopic studies reveal complex surface topography of primary glioma cells and GBM cell lines, which exhibit dense microvilli, membrane folds, filopodia and other membrane protrusions de novo lipogenesis typical for malignant cells It is evident that membrane folds and microvilli along with a typically flattened shape of GBM cells EGFR, CDK4) and deletion of tumor suppressor genes, most frequently PTEN and TP53PTEN and p53 suggests that their combined loss may result in an increased tumorigenic potential Although derived from the same tumor type, glioblastoma cell lines exhibit a wide range of morphological diversity, including fibroblastic, epithelial, glial and other patterns PTEN and p53. In addition to scanning electron microscopy (SEM), we applied the electrorotation (ROT) technique to quantify the area-specific membrane capacity Cm [µF/cm2], related to membrane folding, and the whole-cell capacitance CC [pF], which reflects the total membrane area Cm and CC among GBM lines, revealed by ROT, prompted us to examine in detail the possible signaling pathways involved in the regulation of the cell line-specific membrane morphology.Until now, molecular pathogenesis studies of GBM cells revealed no apparent correlations between morphological and genetic data 2, 37°C) in Complete Growth Medium (CGM), which was either Dulbecco’s modified Eagle’s medium  or minimum essential medium , supplemented with 10% FBS. Mutations of the tumor suppressors PTEN and p53 in the tested cell lines are summarized in The set of 5 human glioblastoma (GBM) cell lines studied here includes DK-MG, GaMG, U87-MG, U373-MG, and SNB19 cells. All cell lines were obtained from ATCC and routinely cultured under standard conditions .Cells were passaged thrice weekly by seeding at about 10For SEM, cells were seeded on microscope cover glass and fixed by addition of 6.25% glutaraldehyde in 50 mM phosphate buffer (pH 7.2) for 10 min at RT and subsequently at 4°C overnight PP amplitude over the frequency range from 100 Hz to 150 MHz. A drop of cell suspension (50–70 µL) was added to the ROT chamber, and a coverslip was placed gently over its center. The cell rotation was observed using a BX50 Olympus microscope . ROT spectra were monitored by decreasing the field frequency in steps (five frequency points per decade). At each field frequency, the rotation speed of lone cells located near the center of the chamber was determined using a stopwatch. The ROT spectra, i.e., frequency dependencies of the ROT speed Ω [radian/s], were normalized to the field strength of 100 V/cm.Electrorotation (ROT) spectra were measured in a microstructured four electrode chamber, described in detail earlier fc1 that induced fastest anti-field cell rotation were performed by the contra-rotating fields (CRF) technique f). The CRF method permits accurate and rapid determination of the maximum rotation frequency fc1, at which ∂Ω/∂f = 0 and the cell stop rotating. In the CRF experiments, 10 µL of cell suspension was added to a four-electrode chamber described previously Measurements of the field frequency N, Ai and fci are the number, magnitudes and characteristic frequencies of the ROT peaks, respectively. Providing that the Maxwell-Wagner relaxation processes are widely separated in the frequency domain, the ROT spectrum of human and other mammalian can exhibit 2–3 peaks in the kHz-MHz range.The ROT spectra of living cells can be presented as a superposition of several Lorentzian curves caused by the Maxwell-Wagner dispersions at the various dielectric interfaces, e.g. at the plasma and nuclear membranes fc1, the cell radius a, the area-specific membrane capacity Cm [µF/cm2] and conductance Gm [mS/cm2] a> is the mean cell radius, σe [µS/cm] is the external conductivity of suspending medium, which is assumed to be held so low that σe<<σi (σi is the intracellular conductivity). The membrane parameters Cm and Gm can be extracted by fitting Eq. 2 to the (fc1·a) data plotted against σe.The theory of single cell electrorotation gives the following relationship between the characteristic frequency of anti-field electrorotation Cm0 ≈ 0.8 µF/cm2, an effective folding factor can be introduced as φ = Cm/C0φ describes the ratio of the actual cell membrane surface area to that of a smooth sphere of the same radius. From the Cm and radius data, the whole-cell capacitance CC [pF] was also calculated as:Given that smooth plasma membrane has a capacitance of Cm value, which represents the membrane capacitance per unit area, the parameter CC accounts for the total electrically accessible cell membrane, including both smooth and folded membrane regions.Unlike the 5 cells/ml was injected into the chamber and the cells were allowed to settle and to adhere to the chamber floor for 10–15 min. The chamber was placed on the stage of a microscope  and the cells were viewed with a 20× objective in transmitted light. The microscope was equipped with a CMOS video camera  connected to the video digitizing board of a personal computer. Images of cells were taken 1–2 min before and at various time intervals of 10 seconds up to 20 min after medium exchange. The cross-section areas of typically 9–10 cells per microscopic field were determined with an image analysis program Image J . At each time interval, the volume (V) of an individual cell was evaluated from its cross-section by assuming spherical geometry. The cell volume was normalized to the original isotonic volume (V0) as: ν = V/V0. The mean ν values (± SE) for a given experiment were calculated from a sequence of ∼160 images and plotted against time.Hypotonic solutions used in the volumetric experiments contained either sucrose or sorbitol as the major osmolyte. The osmolality was adjusted to 50, 100 and 200 mosmol/kg . Cell volume changes were measured by videomicroscopy using a flow chamber designed for rapid exchange of media µg of protein were separated using 4–12% SDS-polyacrylamide pre-cast gels  and transferred to nitrocellulose membranes according to manufacturer’s prescriptions. For protein detection, membranes were incubated with respective primary and species-specific peroxidase-labeled secondary antibodies according to standard protocols. The levels of protein expression were quantified using Image J program and normalized to the β-actin levels.For immunoblot analysis, whole cell lysates were prepared according standard procedures, 20–24 h after splitting the culture. Samples equivalent to 20The primary antibodies used were: rabbit polyclonal anti-PTEN, rabbit polyclonal anti-PI3K p110, mouse monoclonal anti-phospho-AKT (Ser473), rabbit monoclonal anti-phospho-mTOR (Ser2448) , mouse monoclonal anti-p53 , mouse monoclonal anti-Fatty Acid Synthase , mouse monoclonal anti-MDM2 (SMP14) , mouse monoclonal anti-β-actin . Secondary species-specific antibodies for Western blot were labelled with horseradish-peroxidase .The SEM images of adherently growing GBM cells  reveal cAs shown previously Cm [µF/cm2] among 5 GBM lines by means of the contra-rotating field (CRF) technique. To this end, we first analyzed the complete ROT spectra of cells over a frequency range between 1 kHz and 100 MHz. As seen in fc1 of about 10 kHz and two co-field peaks (fc2 and fc3) in the MHz-range. A triple Lorentzian function (Eq. 1) fits very well the ROT spectra  and magnitudes , which are summarized in the The following ROT experiments aimed to compare the area-specific plasma membrane capacitance fc1) is dominated by the capacitive charging of the plasma membrane, whereas the high-frequency co-field peaks (fc2 and fc3) arise from the polarization and/or dielectric dispersions in the cytoplasm and cell nucleus fc1).Previous studies have shown that the low-frequency anti-field peak (e on the plasma membrane peak (fc1). For each cell line, fc1-values and radii (a) of single cells were measured in large samples consisting of 300–420 cells. The cells were suspended in isotonic inositol medium (300 mOsm) of conductivity σe ranging between ∼10 and 50 µS/cm (fc1·a and σe is expected (Eq. 2) and is found in all cell lines .Once the Cm values ranging between 1.88 µF/cm2 in DK-MG and 4.0 µF/cm2 in U373-MG cells . Particularly, the φ values larger than 3 obtained here for cell lines with mutant PTEN or p53 status, or both, are clearly at the upper edge of the φ range measured in 60 tumor cell lines by dielectrophoresis In isotonic medium, the 5 GBM lines exhibited very different MG cells . ExceediCm values of 1.56±0.10 and 2.05±0.12 µF/cm2 obtained, respectively, for HEK293 and HFIB-1 cells, are similar to that of DK-MG cells (1.88 µF/cm2), i.e. the lowest Cm value among 5 tested GBM lines.For comparison, we also analyzed the plasma membrane folding in two non-malignant human cell lines, including the human embryonic kidney HEK293 line and the human fibroblast cell line HFIB-1 (both are adherently growing cell lines). As evident from the Cm values of GBM cells (1.9–4.0 µF/cm2), were really due to excessive membrane folding, we analyzed the impact of hypotonic cell swelling on Cm. The rationale of these experiments was to prove whether, and to what extent, the hypotonic stretching and expected membrane unfolding would cause a reduction of Cm as commonly found in mammalian cells To verify our conclusion that the extremely high isotonic Cm on the external osmolality in all tested GBM lines , in inositol- or sucrose-substituted media. Inspection of the video recordings showed that in all tested cell lines the majority of cells withstood very well the imposed hypotonic stress. Even upon extreme swelling at the lowest osmolality of 50 mOsm , the portions of hypotonically lysed cells were very low: 2–10% DK-MG, 0.5–5% U87-MG, 1–2% GaMG, 1–3% U373-MG, and ∼1% SNB19 cells. These data corroborate the results of ROT measurements  demonstrTo compare in detail the osmotic properties of GBM lines, we analyzed the kinetics of cell volume changes during hypotonic stress. To this end, the cells were rapidly transferred from isotonic CGM (∼300 mOsm) to solutions of different osmolalities  containing sucrose or inositol as the major solute. As seen in the microphotographs , 100-mOsV0 to the Vmax level due to the fast water influx driven by the imposed osmotic gradient. The rates and magnitudes of initial swelling varied widely among the cell lines. DK-MG cells swelled somewhat slower than other cell lines, which is particularly evident from the data obtained in 50- and 100-mOsm sucrose solutions , by applying the Lúcio-model Pw and α values are given in Unlike inositol, the disaccharide sucrose did not permeate the plasma membrane of GBM cells, as evidenced by the ability to RVD over the entire hypotonicity range . The prePw and solutes α , a key enzyme in As seen in As expected, PTEN protein was detected only in DK-MG and GaMG cells which are wild type PTEN We also analyzed the protein levels of fatty acid synthase (FAS). Among 5 GBM lines, DK-MG cells expressed the lowest levels of FAS protein <<U87-MG (2.82) ≈ GaMG (3.17)<<SNB19 (3.71) ≈ U373-MG (4.00).Our ROT measurements of the area-specific capacitance M images , the memBM cells . The CmCm is highly characteristic for a particular cell type or line. Thus, human erythrocytes usually exhibit a Cm of 0.7–0.9 µF/cm2, typical for smooth membranes Cm values of 1.35–1.40 µF/cm2Cm values determined by independent experimental approaches suggests this dielectric quantity as a reliable biophysical marker of a particular cellular phenotype.Numerous studies have shown that Cm data, our ROT measurements provided the values of the whole-cell capacitance CC (pF) for each GBM line. Unlike the area-specific Cm, which reflects membrane folding and microvilli expression, CC takes account of the entire electrically accessible membrane area. As with Cm, we found the CC values to vary greatly among GBM lines, but the rank order of cell lines with respect to CC was completely different: DK-MG (9.4 pF)<U87-MG (15.9)<SNB19 (21.6)<U373-MG (25.3)<GaMG (28.8)  was mainly due to their larger radius (8.5 µm), as compared to DK-MG (6.3 µm). In contrast, the large CC of U373-MG cells (rank 4) was dominated by the extreme membrane folding , rather than by the moderate size (radius = 7.2 µm).In addition to G (28.8) . InteresCC values measured here by ROT compare favorably with the few published CC data of GBM cells obtained in electrophysiological studies. Thus, the mean CC = 15.9 pF of U87-MG cells reported here . The two lines possessing one mutation in either p53 (GaMG) or PTEN (U87-MG), exhibited much higher Cm values, respectively, of 3.2 and 2.8 µF/cm2 (ranks 3 and 2). Interestingly, the cell lines with the highest Cm values (ranks 5 and 4), i.e. U373-MG (4.0 µF/cm2) and SNB19 (3.7 µF/cm2) are mutated in both PTEN and p53. Being twice as high as the Cm of DK-MG cells, the very high Cm values of U373-MG and SNB19 cells corroborate well with the strongly villated membranes of these cell lines revealed by SEM in the present study . The model in i.e. those containing mutations in either p53 or PTEN, or both tumor suppressors , as compared to DK-MG cells (CC = 9.4 pF).Besides the mTOR activation mechanism discussed above, the large variations in membrane area among GBM lines  i.e. the cell line exhibiting the lowest Cm (1.9 µF/cm2) in the present study  may also be involved in the regulation of invasive behavior of GBM cells. Thus, we found recently that two GBM lines U373-MG and SNB19 (mutated in both PTEN and p53), exhibited much higher invasion rates through Matrigel-coated filters (Boyden chamber assay), as compared to DK-MG, GaMG and U87-MG cells . In agreement with this finding, upregulation of PTEN inhibits migration and wound healing properties of glioma cells in vivo.Finally, although the data presented here suggest an apparent correlation between morphological features and PTEN/p53 mutational status as well as lipid metabolism, this study does not definitely prove a causal relationship between genetic features and cellular behavior. Therefore, further studies are warranted to determine if manipulation of PTEN/p53 status and/or lipid metabolism can indeed be directly related to changes in cellular morphology, invasiveness and chemoresistance. Further studies on animal models are also necessary to determine the relationship of these morphological features to behavior of malignant glioma cells Cm. Moreover, Cm was found to correlate with the mutational status of the tumor suppressor genes PTEN and p53. In accordance with dielectric data, SEM showed various membrane extensions in GBM cells, including folds, microvilli and lamellipodia. Although this study involved only established GBM lines, the ROT technique presented here is easily applicable to any malignant cells including those derived from primary tumors. Given that membrane extensions may be implicated in tumor cell motility and metastasis, the dielectric approach presented here provides a rapid and simple tool for screening tumor cell properties in studies on targeting chemo- or radiotherapeutically the migration and invasion of GBM and other tumor cells.The comparative analysis of the dielectric plasma membrane properties revealed a striking difference among 5 established GBM cell lines, most notably, in the degree of membrane folding probed by the area-specific capacitance Figure S1The radius-normalized fc1 values (fc1⋅a) of the indicated GBM lines plotted vs.e. the external conductivity σ The measurements were performed in strongly hypotonic 50-mOsm inositol medium. The lines are best fits of Eq. 2 to the data. The fitted Cm values are summarized in see text and the Legend to (TIF)Click here for additional data file.Figure S2The mean Cm (± SE) values of 5 GBM lines compared by the Student’s t-test, using the Software Origin 8 : (*) denotes P<0.05; n.s. indicates that the difference was not significant (P>0.05). The differences in Cm between GBM lines were statistically significant, except for the pair U373-MG vs. SNB19 cells, i.e. the two cell lines mutated in both PTEN and p53.(TIF)Click here for additional data file.Figure S3Determination of the area-specific membrane capacitance of two non-cancerous, adherently growing cell lines, including the human embryonic kidney HEK293 cells and the human fibroblast cells HFIB-1. The measurements were performed in isotonic 300-mOsm inositol medium. The fc1 data were obtained by the CRF-technique. Each symbol is the mean (±SE) from 16–20 cells measured at closely similar conductivities. The lines are best fits of Eq. 2 to the CRF data sets, containing ∼300 HEK293 cells and ∼400 HFIB-1 cells. The fitted Cm values are 1.56±0.10 and 2.05±0.12 µF/cm2 respectively, for HEK293 and HFIB-1 cell lines.(TIF)Click here for additional data file.Figure S4Boyle van’t Hoff plots for the indicated GBM cell lines. Each data point represents the mean νmax value . The data were fitted by the Boyle van’t Hoff equation: conc is the solution osmolality, the isotonic osmolality is conciso = 300 mOsm, the term β represents the osmotically inactive volume fraction at 300 mOsm. From the Y-intercepts, the β values were found for each cells and summarized in (TIF)Click here for additional data file.Table S1Best-fit parameters of the Lorentzian function (Eq. 1) to the ROT spectra of 5 GBM cell lines, such as shown in.(DOCX)Click here for additional data file.Table S2Cell radius and dielectric properties of GBM cells in strongly hypotonic medium of osmolality 50 mOsm. The Cm and CC values were derived from the data shown in (DOCX)Click here for additional data file.


Interestingly, we observed that the basal protein expression levels of EMT markers show a significant correlation with cell viability in response to elisidepsin treatment in a panel of 12 different breast and pancreatic cancer cell lines. In addition, we generated three elisidepsin treatment-resistant cell lines  and analyzed the pattern of expression of different EMT markers in these cells, confirming that acquired resistance to elisidepsin is associated with a switch to the EMT state. Furthermore, a direct correlation between basal HER3 expression and sensitivity to elisidepsin was observed; moreover, modulation of HER3 expression levels in different cancer cell lines alter their sensitivities to the drug, making them more resistant when HER3 expression is downregulated by a HER3-specific short hairpin RNA and more sensitive when the receptor is overexpressed. These results show that HER3 expression is an important marker of sensitivity to elisidepsin treatment.Elisidepsin  is a new synthetic depsipeptide, a result of the PharmaMar Development Program that seeks synthetic products of marine origin-derived compounds. Elisidepsin is a drug with antiproliferative activity in a wide range of tumors. In the present work we studied and characterized the mechanisms associated with sensitivity and resistance to elisidepsin treatment in a broad panel of tumor cell lines from breast and pancreas carcinomas, focusing on different factors involved in epithelial-mesenchymal transition (EMT) and the use of HER family receptors in predicting the  Elysia rufescensElisidepsin , a synthetic cyclic peptide originally isolated from the marine mollusk in vitro studies identifying HER3 and the downstream signaling pathway PI3K-AKT as major determinants of the cytotoxic activity of elisidepsin One of the several targets that are proposed to be involved in the cellular response to elisidepsin treatment is the human epidermal growth factor receptor family (HER) with several On the other hand, specifically in relation to epithelial cells, one of the best-described processes that affects the composition of the cell membrane is that of the epithelial-mesenchymal transition (EMT), which is where cells downregulate their cell-cell junctions and acquire spindle cell morphology In most cases, this is associated with transcriptional repression of E-cadherin In this scenario, until the proposed above targets are validated, robust models that permit the identification novel predictive biomarkers are essential. To this end, and due to the increasing evidence supporting a role for the EMT in the progression of many cancer types, with critical roles in invasion and metastatic dissemination, we decided to study both HER3 and EMT as new predictive markers of elisidepsin treatment sensitivity in a panel of breast and pancreatic cell lines.In this report, we show that continued exposure to elisidepsin is correlated with a downregulation of epithelial markers in four different cancer cell types . This behavior is further accompanied by several morphological and signaling changes, resulting in the upregulation of mesenchymal markers. Furthermore, we investigated the effect of the drug on the expression of HER proteins and systematically compared the elisidepsin sensitivity of cell lines overexpressing and knocking-down HER3 receptor. Finally, we identified HER3 expression as the most important sensitivity marker of elisidepsin studied.50) values for elisidepsin, as measured by a crystal violet assay using a spectrophotometer, ranged from 0.075 to 14 µM within the cell line panel (We performed cell viability assays in a panel of 12 cell lines (6 breast cancer cell lines and 6 pancreatic carcinoma cell lines) to determine if there was a correlation between epithelial or mesenchymal expression markers and cell sensitivity to elisidepsin. Cells were treated with increasing concentrations of the compound for 72 h. The half maximal (50%) inhibitory concentration . The other cell lines were classed as being sensitive to the drug .According to the results of a previous paper from our lab and others 50 values was seen by Sewell et al. 50) the cells grew more slowly than the parental ones  in a panel of 12 cell lines. The protein expression of E-cadherin, β-catenin, vimentin, Slug, Snail and Twist-1 were assessed by immunocytochemical and western blot analysis, while the protein expression of E-cadherin, β-catenin, and vimentin were evaluated by immunohistochemical analysis.We aimed to determine whether the various elisidepsin-sensitive cancer cell lines shared similar basal levels of EMT genes. In the breast cancer cell lines we found E-cadherin expression in the sensitive cell lines. All cell lines had detectable expression of β-catenin, whereas Slug expression was variable and not related to their sensitivity to elisidepsin. Furthermore, Snail expression was only found in MDA-MB-435, and all the cell lines that exhibited levels of Twist-1 and vimentin were less sensitive to the drug . In contThe primary mechanisms of action of elisidepsin remain to be elucidated but we and other groups have found that after 4 h treatment with 1 µM elisidepsin, HER3 receptor levels are downregulated in a panel of different cell lines, including lung, breast, melanoma and colon carcinomas 50. Elisidepsin concentration was increased every week until cells became resistant to the drug, after approximately 12 months in the case of MCF-7, and after approximately 4 months in the case of HPAC and AsPC-1. The morphology of the resistant cancer cell lines was modified after continuous exposure to the drug when compared to that of the parental cell lines (data not shown). Our hypothesis was that the loss of epithelial markers observed in our panel of cancer cell lines could be responsible for the resistance to elisidepsin treatment, which would in turn result in the acquisition of mesenchymal markers in these cells.Three elisidepsin-resistant cancer cell lines [one breast (MCF-7) and two pancreatic ] were generated by continuous exposure to increasing concentrations of the drug . Cancer cell lines were exposed to elisidepsin at a starting concentration of its ICWe then performed western blot analysis of the cancer cell lines with acquired resistance and compared them to the corresponding parental control cells. We identified that the three different cancer cell types with acquired resistance to elisidepsin had altered basal levels of EMT markers . All resWe also performed the same approach in different resistant cell lines from colon and lung  with similar results . AnalysiFurthermore, we wanted to see if these cells also showed different expression levels of HER family members and proteins of their signaling pathways. We observed that the levels of all HER family members and their downstream signaling partners were downregulated in all resistant cancer cell lines 4. A suppBased on previous studies from our group and others demonstrating that elisidepsin downregulates the HER3 receptor tyrosine kinase and that high expression of HER3 is prevalent in a broad number of different tumor cells, we investigated if modulation of protein expression levels of the HER3 receptor affects sensitivity to elisidepsin in a panel of tumor cell lines with variable expression of this receptor.To examine this experimentally, we utilized a shRNA construct to stably reduce HER3 expression in a panel of cell lines . Stable To investigate whether ectopic HER3 expression affects the elisidepsin sensitivity of low HER3-expressing cells, the levels of HER3 were increased by transfecting cells with a cDNA encoding HER3, which resulted in increased sensitivity of the cells to elisidepsin. In comparison to control cells transfected with a LUC vector, decreased cell viability was noted in HER3-transfected cells . AltogetElisidepsin is a novel marine compound with a potent cytotoxic activity in various tumor cell lines. The mechanisms of actions of this compound remain poorly understood, although several targets have been proposed to be involved in the cellular response to elisidepsin treatment, such as fatty acid-containing ceramides, fatty acid 2-hydroxylase (FA2H), lysosomes, lipid rafts and epithelial growth factor receptors, including the HER receptors In the present study we explored whether basal levels of EMT markers and HER receptor proteins could be predictive markers for elisidepsin treatment. The role of the cell membrane as an important target of elisidepsin was studied in breast and pancreas cancer cell lines. Basal levels of EMT protein expression markers showed a significant correlation with the cell viability response to elisidepsin treatment in a panel of 12 different cancer cell lines. The epithelial marker E-cadherin protein was significantly expressed in the sensitive cell lines (p  = 0.0364) while expression of the mesenchymal markers vimentin, Twist-1 and Snail, was found in all cell lines with reduced sensitivity to the drug.Furthermore, this study showed that continuous exposure to elisidepsin correlates with a downregulation of epithelial markers in 4 different cancer cell types . Loss of epithelial markers was further evidenced by the detection of morphological changes in the cells. These changes, which were observed after continuous long-term exposure of different cell types to elisidepsin, suggest that the drug is able to modify the composition of the plasma membrane. This behavior was further accompanied by signaling changes, resulting in the upregulation of mesenchymal markers. This analysis confirmed that acquired resistance to elisidepsin is associated with a switch to the EMT state.On the other hand, regarding HER family receptors, we observed an association between HER3 protein expression and sensitivity to elisidepsin treatment in a variety of cell lines (p  = 0.0091). The other members of the HER family were also checked by western blotting and we did not find any significant correlation. Interestingly, HER4 expression was observed in 4 out of 5 elisidepsin-sensitive breast cancer cell lines, and further studies that include more breast cancer cell lines are necessary to establish the potential predictive marker of the HERs for elisidepsin sensitivity in breast cancer models. Cell lines that were less sensitive to elisidepsin had lower or undetectable levels of HER3 in comparison with sensitive cell lines, supporting a hypothetical role for HER3 in the cellular response to this drug, although other authors propose that HER3 is part of a secondary process involving cell membrane alterations due to elisidepsin treatment Malignant cells commonly possess overactive signal transduction cascades that provide potential selective targets for antitumor drugs In summary, we present solid evidence that sensitivity to elisidepsin correlates with HER3 receptor expression. However, it remains to be elucidated why elisidepsin affects HER3 and why its effects depend on HER3 expression.Elisidepsin  was obta2.Cell lines were obtained from the American Type Culture Collection . The following cell lines were maintained in RPMI 1640 with 4 mM L-glutamine: MDA-MB-435, MDA-MB-231, MCF-7 and SKBR3 (breast carcinoma), and AsPC-1 and BxPC-3 (pancreas carcinoma). The following cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) with 4 mM L-glutamine and 4.5 g/L glucose: HCT116 (colon carcinoma), MDA-MB-468 (breast carcinoma), and HPAC, PANC-1 and MiaPaCa-2 (pancreas carcinoma). A549 (lung carcinoma) was maintained in Ham's F-12 medium supplemented with 1 mM L-glutamine. Finally, DMEM:Ham's F12 (1∶1 mixture) supplemented with 1 mM L-glutamine was used to maintain BT474 (breast carcinoma). HPAC resistant to elisidepsin 8 µM and AsPC-1 resistant to elisidepsin 4.5 µM were generated by continuous exposure to the drug for ∼4 months, while HCT 116 resistant to elisidepsin 100 µM, A549 resistant to elisidepsin 25 µM, and MCF-7 resistant to elisidepsin 4 µM were generated by continuous exposure to the drug for ∼1 year. The media for all cell lines were supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin and 10 mM HEPES and were cultured in a 37°C humidified atmosphere containing 95% air and 5% COImmediately prior to use in western blotting, cultured cells were lysed and collected in lysis buffer. Lysates were centrifuged and supernatants were collected for protein concentration determination using the Bradford  method. Equal amounts of protein were separated by 8% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels, electrophoresed at 100 V and electroblotted onto polyvinylidene difluoride membranes  at 0.4 A at room temperature. Blots were blocked in 5% nonfat dry milk in phosphate-buffered saline (PBS) for 1 h at room temperature. After blocking, membranes were incubated overnight with primary antibodies against HER1 ; HER2 ; HER3 ; HER4 (111B2), Akt (#9272), γ-catenin (#2309) and Snail ; MAPK (C-14), Slug (H-140) and Twist-1 ; β-actin ; vimentin ; and E-cadherin and β-catenin . After incubation with horseradish peroxidase-conjugated secondary antibodies, antigen-antibody complexes were visualized using enhanced chemiluminescence . Western blots were repeated in independent conditions at least twice and representative blots are shown. Densitometrical quantification of autoradiograms was performed using ImageJ software  by normalizing to the intensity of β-actin in each sample and are expressed in arbitrary densitometric units.Cells were seeded on coverslips at 60% confluence, fixed in 4% formaldehyde/PBS, permeabilized in 100% methanol for 20 min, and blocked in 2% BSA for 1 h. Fixed cells were incubated with anti-E-cadherin  and anti-β-catenin , anti-Snail , anti-Vimentin , and anti-Slug and anti-Twist-1  overnight at 4°C. After washing with PBS, cells were incubated for 1 h in blocking buffer at room temperature with either 1∶800 Alexa Fluor 546 rabbit anti-mouse IgG  or 1∶200 Alexa Fluor 647 mouse anti-human (Invitrogen). Cells were washed twice with PBS and once with distilled water. Finally, cells were mounted in Citifluor  before observation and analysis with fluorescence microscopy.IHC using the avidin-biotin-peroxidase technique was performed for each antibody. Five-micron-thick sections were cut from formalin-fixed, paraffin-embedded cell pellets and placed on poly-L-lysine-coated glass slides. Sections were deparaffinized in xylene and rehydrated in graded alcohol. Endogenous peroxidase was blocked by immersing the sections in 0.1% hydrogen peroxidase in absolute methanol for 20 min. For antigen retrieval, the tissue sections were heated in a pressure cooker in 10 mM citric acid monohydrate, pH 6.0, for 5 min, and then incubated with primary antibodies for 60 min at room temperature. IHC was performed with the Benchmark XT slide stainer . The primary antibodies and dilutions used were: anti-HER3 , anti-E-cadherin , anti-β-catenin  and anti-vimentin . All slides were hematoxylin counterstained, dehydrated, and mounted. Negative controls were performed by omitting the primary antibody and showed minimal non-specific signal.Cells were plated overnight at a density of 50,000 cells/well. Cell lines were treated with various concentrations of elisidepsin for 72 h. At least 3 wells were used for each condition and cell viability was measured by a crystal violet assay. Briefly, cells were fixed after each treatment in 1% glutaraldehyde for 20 min, washed twice in PBS, stained with 0.1% crystal violet for 30 min and then washed with abundant deionized water. Colorant was recovered using 5% acetic acid and optical density was measured at 590 nM with an ELISA plate reader.The pIRES-HER3 and the pIRES-Luciferase (LUC) were kindly donated by Dr. Scaltriti . The pIRES-LUC was used as a control for transfection. The pIRES vectors confer hygromycin resistance. Cells were transfected for 12 h with Jet Pei . To eliminate untransfected cells and generate stably expressing HER3 cell lines, medium supplemented with hygromycin (Sigma-Aldrich) was added 24 h after transfection, and cells underwent selection for 10 days.GATCCAAGAGCGACTAGACATCAAGCTTCAAGAGAGCTTGATGTCTAGTCCCTCTTTTTTTACGCGTG, shHER3_3.1R: AATTCACGCGTAAAAAAAAGAGCGACTAGACATCAAGCTCTCTTGAAGCTTGATGTCTAGTCGCTCTTG; shHER3_3.3F: ATCCGCCAATACCAGACTGTACTTCAAGAGAAGTACAGTGTCTGGTATTGGTTTTTTACGCGTG, and shHER3_3.3R: AATTCACGCGTAAAAAACCAATAC CAGACACTGTACTCTCTTGAAGTACAGTGTCTGGTATTGGCG. The different sequences were cloned into the lentiviral vector pLKO.1 (Sigma-Aldrich). The pLKO.1-shRNA LUC was used as a control for transfection. All vectors encode puromycin resistance. Plasmids pVSVG and pCMVΔR8.91 for the expression of packaging and envelope proteins were kindly provided by Dr. Peeper . Two plates seeded with 1.5×106 HEK 293T cells were co-transfected in DMEM 10% FBS with 2 µg of pLKO.1, 2 µg of pCMVΔR8.91 and 2 µg of pVSVG and incubated overnight. Cells were washed and incubated in 10% CO2 with medium containing 5% FBS. After 48 h, the virus-containing supernatant was recovered and filtered with 0.45 nM filters .Short hairpin RNAs (shRNAs) were used for inhibiting HER3 expression in different cancer cell lines. The following sequences were used: shHER3_3.1F: Titration was performed by infecting cells with the recovered viral particles in the presence of 4 µg/mL polybrene (Sigma-Aldrich). Western blot was used to assess the silencing efficacy of the two shHER3 sequences, and the most effective one (shHER3.3) was chosen. To obtain cell lines with stable depletion of HER3, infected cells were selected with puromycin for three days.50≤1 µM) and Less Sensitive (includes cell lines with an elisidepsin IC50>1 µM). Cell growth data are expressed as the mean ± standard deviation (SD). Statistical significance was set at a two-tailed p value of 0.05.Statistical studies were performed with the Statistical Package for the Social Sciences . The Mann-Whitney test was used to find associations of the parameters analyzed between two previously selected groups, Sensitive  Cells were treated with 1 µM of elisidepsin for 4 h, the culture medium was changed and cells were maintained in the fresh medium for 4, 24, 48 and 72 h. HER1-4 protein expression levels were analyzed by western blot using 50 µg of protein from total MCF-7 cell lysates loaded in SDS-PAGE gels. Membranes were stripped and reprobed with anti-β-actin to verify equal protein loading. B) Cells were treated with 1 µM of elisidepsin for 4 h and proliferation was measured by a crystal violet assay at different time points (white squares) and compared to untreated cells (black diamonds). Results are expressed as the mean ± SD of two independent experiments. C, control.(TIF)Click here for additional data file.Figure S2Statistical analysis of EMT basal expression levels of breast and pancreas cancer cell lines. Levels of ErbB3 protein were quantified using western blot analysis  by densitometry. The graph represents the relative ErbB3 expression in elisidepsin-sensitive (IC50≤1 µM) and -resistant (IC50>1 µM) cell lines. The Mann-Whitney test showed a statistically significant p value of 0.015.(TIF)Click here for additional data file.Figure S3Elisidepsin cell sensitivity is associated with HER3 expression levels. Levels of HER1, HER2, HER3 and HER4 protein were quantified with western blot analysis  but not with the other members.analysis  and subs(TIF)Click here for additional data file.Figure S4Generation and characterization of elisidepsin-resistant cell lines from colon and lung. A) Cells were lysed, proteins were extracted and western blots performed with an equal amount of cell lysate (50 µg protein). Expression of epithelial - and mesenchymal -associated proteins differentiates between elisidepsin-sensitive and elisidepsin-resistant cell lines. β-actin was used as an internal control. These western blots were performed in triplicate. B) Expression levels HER1, HER2, HER3, HER4, pAkt, and pMAPK were analyzed by western blot using 50 µg of protein cell lysate. The membranes were stripped and reprobed with anti-β-actin to verify equal protein loading. HCT 116 (C) and A549 (D) elisidepsin-sensitive cancer cell lines were rendered resistant by persistent exposure to increasing concentrations of elisidepsin. Cells were treated with elisidepsin at the indicated concentrations for 72 h and cell viability was measured using a crystal violet assay. Error bars show the SD of three replicate experiments. C, control; R, resistance.(TIF)Click here for additional data file.Figure S5Chemical structure of elisidepsin.(TIF)Click here for additional data file.


While some phenotypes of mice lacking lncRNAs reveal potential targets for treating diverse human diseases, others do not match the expectations from experimental manipulations in cell lines reported over the last 10 years. In effect, it has become clear that principles learned about lncRNA functions in cell lines can be very different when tested in animal models  regulate transcription of the Ubx1 homeodomain transcription factor  were indoctrinated regarding the importance of in vivo studies. Thankfully, Terry Grodzicker, the editor at Genes and Development did not share the Nature editor's views, and agreed to send our paper out for peer review. This led to publication of our work on Evf2 trans-acting activity in 2006  a few years later indicated lncRNA repression in cis  and repressor (MECP2), and through anti-sense regulation represses adjacent gene expression , previously thought to be critical for nuclear speckles and splicing, show no morphological alterations  mice and in mice treated with anti-sense oligos to MALAT1  rescues enhancer methylation, but not cis gene expression effects  , and demonstrates the power of genetics and lncRNA biology when combined  lncRNA directs cardiovascular lineage commitment in embryonic stem cells (Klattenhoff et al., in vitro and in vivo studies are likely to be most powerful when generated in the right system for the right purpose.While the arguments for utilizing mouse models to study lncRNA mechanism and significance are clear, there are several arguments, in addition to the discovery argument, to continue studies in cell lines. For instance, in the field of regenerative medicine, lncRNAs have the potential to guide human or mouse embryonic stem cells toward specific lineages, or reprogram induced pluripotent stem cells. Work on lncRNAs controlling retinal fate specification in mice in vivo described above as a biological anomaly, such differences are not specific to lncRNA studies. The REST conditional mouse knockout serves as a salient example of how a whole field can be surprised and challenged when a key in vivo experiment refutes previous dogma (Aoki et al., in vivo.Although one may dispel the differences between lncRNA activities in cell lines and in vivo models is not only important to grant reviewers, NIH program officers, the editors of some journals, and human disease, but it is important for answering questions that eventually establish the basic principles in the field. In the case of modern lncRNAs, mechanistic studies in cell lines have so far outweighed studies in mice. However, multiple in vivo models are shaking up some of the previous lncRNA dogma, revealing lncRNA biological significance and functional diversity, as well as guiding the future of the lncRNA field.Determining biological significance using


Genetically engineered T-cells that express chimeric antigen receptors (CARs) to directly target tumor-expressed antigens have shown remarkable activity in clinical trials for hematologic malignancies, but remain unproven for the treatment of solid tumors. We find that in addition to its well known high expression in neuroblastoma, the disialoganglioside GD2 is also highly expressed on pediatric sarcomas. Some human sarcoma cell lines have GD2 expression levels equivalent to that of the prototypical neuroblastoma cell line LAN5, and immunohistochemical staining of primary human tumor tissue samples taken from essentially all patients with metastatic osteosarcoma and a subset of patients with alveolar rhabdomyosarcoma demonstrate robust expression of GD2 on the cell surface. Based on these results, we have developed models to test the ability of GD2-CAR T-cells to target and lyse human sarcomas in vitro and in vivo. We find that GD2-CAR modified T-cells induce specific lysis of GD2-expressing solid tumors in vitro even at effector:target ratios as low as 1:1, but fail to induce a significant response in vivo using the same human cell lines. Interestingly, we discovered that the pediatric sarcoma xenografts induce a large expansion of murine CD11b+Gr1+ myeloid-derived suppressor cells (MDSC) that inhibit human T-cell responses in vitro. These results lead us to adopt a combinatorial therapeutic strategy in which we first neutralized the suppressive potential of MDSC by administration of all-trans retinoic acid (ATRA) followed by GD2-CAR therapy. This combinatorial approach results in significant improvements in both overall survival and tumor growth. Given that retinoids are already available in the clinic, these results suggest that the effectiveness of CAR T-cell therapy for solid tumors could be enhanced by coadministration of retinoids to modulate the myeloid derived suppressor cells.


Two human γ-herpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), are putative etiologic agents for Burkitt lymphoma, nasopharyngeal carcinoma, and some cases of gastric cancers, and Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma (PEL) especially in AIDS setting for the latter case, respectively. Since such two viruses mentioned above are highly species specific, it has been quite difficult to prove their oncogenic activities in animal models. Nevertheless, the viral oncogenesis is epidemiologically and/or in vitro experimentally evident. This time, we investigated gene expression profiles of KSHV-oriented lymphoma cell lines, EBV-oriented lymphoma cell lines, and T-cell leukemia cell lines. Both KSHV and EBV cause a B-cell-originated lymphoma, but the gene expression profiles were typically classified. Furthermore, KSHV could govern gene expression profiles, although PELs are usually coinfected with KSHV and EBV.Among herpesviruses,  Several viruses could induce cancers in human beings. For examples, some papilloma viruses (PVs) should be etiologic agents for cervical cancers , hepatitChronic inflammation caused by these viruses should be important factors, but it is not forgettable to keep in our minds that such inflammation itself is primarily caused by the viral infection . Except γ-herpesviruses such as EBV and KSHV are DNA viruses and do not have the genome integration process in their life cycles and just present as episomes in the infected nuclei for lives after establishing latent infection, since their genomes replicate and are partitioned according to the host cell cycles by utilizing host cellular replication machinery 31], BCBLAs mentioned above, there are three definite diseases caused by KSHV. They are KS, PEL, and MCD. Especially in AIDS setting, KSHV has a very tight link with these diseases, and that is the virulence of KSHV emerges under the condition. It seems to be meaningful to know gene expression profiles of tumors, since such gives us information about origin of tumor cells, mechanism of tumorigenesis, designs of treatment, and so on. And thus; several reports have been published .PROX1, a master regulator of lymphatic development and downregulation of blood vascular genes, in infected human dermal microvascular endothelial cells (HDMECs) [PROX1. These findings suggest that KSHV infection should induce a comprehensive reprogramming of blood vascular endothelial cells (BECs) to adopt a lymphatic endothelial cells (LECs). In the tissues of KS, a kind of cytokine and interleukin-6 (IL-6), basic fibroblast growth factor (bFGF), tumor necrosis factor-α (TNF-α), oncostatin M, interferon-γ (IFN-γ), and so on storm happens. In in vitro KSHV infection study; however, IL-6, oncostatin M, TNF-α, and IFN-γ inductions were not induced. In contrast, tumor growth factor β1/β3 and TGFb R2, CCL5 [The cellular origin of the spindle cells of KS is poorly defined and could be originated from vascular endothelial cells and various kinds of cytokines, chemokines, and growth factors are expressed , 36. A r(HDMECs) , 38. KSHR2, CCL5 , CCL8 , interleukin-15 receptor α subunit precursor (U31628), interferon-induced 56-kd protein , and protein-tyrosine phosphatase 1C (PTP1C) SHP1 (X62055) and HLA class II histocompatibility antigen α chain (K01171) and HLA-DR antigen-associated invariant subunit (X00497) were prominently induced by the factor of ten or more [in vitro. Most of BL, however, show latency I phenotype, and thus; this experiment model reflects LCL rather than BL [− Akata [− Akata and EBV+ Akata were stimulated with IgG crosslinking, and lytic replication was induced. They analyzed cellular gene expression as well as viral gene expression. In this case, data did not reflect effects of EBV on BL, since this was just lytic replication/reactivation process, and almost all viral genes were expressed, which presumably took a substantial effect on cellular gene expression.EBV is another human oncogenic nd so on . BL is aangement , 51. Stu or more . High-mo or more . In thisursor U3128, inter[− Akata . EBV− AkWe recently investigated gene expression profiles of PEL cell lines, comparing with those of the other uniquely categorized cell lines, one of which was BL cell lines with or without EBV infection and another of which was T-cell leukemia cell lines (TCLs) . All PELOur obtained results were that three kinds of lines were typically classified into respective groups. Although the results might reflect just differentiation status of these cell lines, KSHV would never be lost from the PEL cell lines and BC1 coinfected with KSHV, and EBV was classified into the PEL cell category, suggesting that KSHV should be more dominant in gene expression control. Among about thirty thousand genes analyzed this time, we could extract sixty-three genes typically higher in BL cell lines and also sixty genes predominantly higher in PEL cell lines. For example, CD79A (NM_001738) and B (NM_000626), which are components of B-cell receptor and contain cytoplasmic immunoreceptor tyrosine-based activation motifs were highly expressed in BL without doubt. Accordingly, BCR downstream signaling 1  was also higher in BL cell lines. A mature B cell-marker, CD22 (NM_001771), was characteristically expressed in BL not in PEL. Among very highly expressing genes in PEL, we found methyl CpG-binding domain protein , interleukin 2 receptor beta , and angiopoietin 1 . Such gene expression in PEL might suggest cellular environment and pathophysiologic status in the patient bodies under immunosuppression due to AIDS established by human immunodeficiency virus 1 (HIV-1) and KSHV infection.Cip1/WAF1 (NM_000389), IL15 receptor α (NM_002189) and HLA-DR , which are reported to increase by EBV infection, this gene expression was indeed higher in B cell originated PEL and BL with a few exceptions in our analysis , B-cell specific markers: PAX 5 (NM_016734), Oct2 (XM_068123), and a GC B-cell marker: BCL6 (NM_001706) were higher in BL cell lines and preplasma cell markers; IRF4/MUM1 (NM_002460) and PRDM1/BLIMP1 (NM_001198) are definitely higher in PEL cells, assuring that BL should be derived from GC B cell and PEL from post-GC plasmablasts , Table 2If there are common genes in all tumor cell lines analyzed this time, such genes could be generally required for their establishment and/or maintenance. Thus, we mined the data in such point of view and found a couple of genes were commonly overexpressed compared to normal PBMC , Table 3In the same way, we also mined the data to find less expression in all types of cell line , which mStudying gene expression profiles gives us various kinds of information. The analysis especially in cancer will lead to understanding how cancers are generated and maintained and to design what to do in order to suppress cancer growth. It is, however, just screening, and we have much work to do for this aim.


Lafoensia pacari is a native species from South America which has been used in traditional medicine as anti-ulcerogenic and anti-inflammatory for several diseases, but the antineoplastic potential still have not been elucidated so far, though its etnopharmacological indication. The aim of this study was to evaluate the anti-neoplastic effect of L. pacari ethanolic extract in three human lung neoplastic cell lines.in vitro, being two originally from non-small cell lung carcinoma (A549 and H2023) and one of giant cell lung carcinoma obtained from pleural effusion (H460). Cells were grown in 96 well plates under regular cell culture condition and treated with different concentrations of L.pacari ethanolic extract, then analyzed using 3--2,5-diphenyl bromide tetrazolium, widely used to determine the viability of cultured cells. The IC50 value and the regression curve were calculated with 5.0 Prism . Also, the Caspase-3 Assay Kit for Live Cells  was used to determine the mode of action of L. pacari at IC50 concentration.For the assessment of cytotoxicity, cell lines were grown L.pacari, we determined a dose-dependent effect of L. pacari extract in all cell lineages, being the H460 cell line the most sensitive by means of lowest IC50. Thus, we also showed that this effect was due to induced caspase-3 dependent apoptosis.After the treatment for 72h with the ethanolic extract of L. pacari demonstrated an antineoplastic effect in all cell lines. The Caspase-3 activation in tumor cells after treatment with L. pacari suggests that the cytotoxic effect is related to activation of the intrinsic apoptotic pathway, leading ultimately to cell death. Further studies are under investigation to determine the specific substances responsible for these effects.The extract of CAPES and FAPESP (2008/56584-2).


All patients received immunoselected Tregs   and 4 days later positively immunoselected CD34+ cells  together with Tcons . CD4/CD25+ Tregs (purity 92.7 ± 2.1) consisted of 33.6% ± 13.1 CD25high; 58.1% ± 6.6 CD25int; 5.8% ± 2.5 CD25low; 65.7% ± 11.8 FoxP3; 17.4% ± 7.2 CD127 (mean ± SD). No GvHD prophylaxis was administered. 26/28 patients engrafted. No GvHD developed in 24/26 patients, 2 developed ≥ grade II GvHD. Ten patients died . CD4 and CD8 counts reached, respectively, 50/μL medianly on days 34 (range 19-63 days) and 24 (range 15-87); 100/μL medianly on days 47 (range 28-100 days) and 34 (range 19-95); 200/μL on days 70 (range 41-146 days) and 61 (range 21-95). A wide T-cell repertoire developed rapidly with high frequencies of specific CD4+ and CD8+ for opportunistic pathogens. Episodes of CMV reactivation were significantly fewer than after our "standard haplo" transplants. In KIR ligand-mismatched transplants, speed of NK cell reconstitution/maturation and size of donor vs recipient alloreactive NK cell repertoires were preserved. In conclusion, in the setting of haploidentical transplantation infusion of Tregs makes administration of a high dose of T cells feasible for the first time. This strategy provides a long-term protection from GvHD and robust immune reconstitution.Haploidentical transplantation, with extensive T cell depletion to prevent GvHD, is associated with a high incidence of infection-related deaths. The key challenge is to improve immune recovery with allogeneic donor T cells without triggering GvHD. As T regulatory cells (Tregs) controlled GvHD in preclinical studies, the present phase I/II clinical trial evaluated the impact of early infusion of donor CD4/CD25+ Tregs, followed by an inoculum of donor mature T cells (Tcons) and positively immunoselected CD34+ cells. Twenty-eight patients  were enrolled from September 2008 onwards; 22 had AML , 5 had ALL  and 1 had high grade NHL in relapse. Conditioning was: 8 Gy single fraction TBI, thiotepa (4 mg/kg × 2), fludarabine (40 mg/m