Characterization of human embryonic stem cells with features of neoplastic progression
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Veronica Ramos-Mejia
PROVIDER: E-GEOD-13995 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
Characterization of human embryonic stem cells with features of neoplastic progression.
Werbowetski-Ogilvie Tamra E TE Bossé Marc M Stewart Morag M Schnerch Angelique A Ramos-Mejia Veronica V Rouleau Anne A Wynder Tracy T Smith Mary-Jo MJ Dingwall Steve S Carter Tim T Williams Christopher C Harris Charles C Dolling Joanna J Wynder Christopher C Boreham Doug D Bhatia Mickie M
Nature biotechnology 20090104 1
Cultured human embryonic stem (hES) cells can acquire genetic and epigenetic changes that make them vulnerable to transformation. As hES cells with cancer-cell characteristics share properties with normal hES cells, such as self-renewal, teratoma formation and the expression of pluripotency markers, they may be misconstrued as superior hES cells with enhanced 'stemness'. We characterize two variant hES cell lines (v-hESC-1 and v-hESC-2) that express pluripotency markers at high levels and do not ...[more]
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Project description:Cultured human embryonic stem (hES) cells can acquire genetic and epigenetic changes that make them vulnerable to transformation. As hES cells with cancer-cell characteristics share properties with normal hES cells, such as self-renewal, teratoma formation and the expression of pluripotency markers, they may be misconstrued as superior hES cells with enhanced ‘stemness’. We characterize two variant hES cell lines (v-hESC-1 and v-hESC-2) that express pluripotency markers at high levels and do not harbor chromosomal abnormalities by standard cytogenetic measures. We show that the two lines possess some features of neoplastic progression, including a high proliferative capacity, growth-factor independence, a 9- to 20-fold increase in frequency of tumor initiating cells, niche independence and aberrant lineage specification, although they are not malignant. Array comparative genomic hybridization revealed an amplification at 20q11.1-11.2 in v-hESC-1 and a deletion at 5q34a-5q34b;5q3 and a mosaic gain of chromosome 12 in v-hESC-2. These results emphasize the need for functional characterization to distinguish partially transformed and normal hES cells.
2008-12-25 | GSE13995 | GEO
Project description:Progression to androgen independent is the main cause of death in prostate cancer, and the mechanism is still unclear. By reviewing the expression profiles of 26 prostate cancer samples in a holistic view, we found a group of genes differentially expressed in androgen independent compared with androgen dependent groups (p value< 0.01, t test). Focusing on apoptosis, proliferation, hormone and angiogenesis, we found a group of genes such as thioredoxin domain containing 5 (TXNDC5), tumor necrosis factor receptor superfamily, member 10a (TNFRSF10A), ribosomal protein S19 (RPS19) and Janus kinase 2 (JAK2) up-regulated in androgen independent prostate cancer, which could play important roles in the transition from androgen dependent to androgen independent and could be biomarkers of prognosis. The main aim was comparing the androgen dependent and androgen independent prostate cancer to identify differentially expressed genes. In addition, we added several normal prostate tissue sample for comparisons. Totally 29 experiments were performed without replicates. 3 for normal prostate tissue, 8 for androgen independent cancer and 18 for androgen dependent prostate cancer. In all experiments, the reference samples are common reference, a pool with unrelated fetal tissues.
2007-09-09 | E-GEOD-5803 | biostudies-arrayexpress
Project description:Copy number variations (CNVs) account for a substantial proportion of human genomic variation, and have been shown to cause neurodevelopmental disorders. We sought to determine the relevance of CNVs to the aetiology of schizophrenia. Whole genome, high resolution, tiling path BAC array comparative genomic hybridization (array CGH) was employed to test DNA from 91 individuals with DSM-IV schizophrenia. Common DNA copy number changes that are unlikely to be directly pathogenic in schizophrenia were identified by comparison to a reference dataset of 372 control individuals analysed in our laboratory, and a screen against the Database of Genomic Variants. The remaining aberrations were tested for inheritance from the parents, and validated with Affymetrix 250K SNP arrays or 244K Agilent oligo-arrays. Thirteen aberrations satisfied our criteria. Two of them are very likely to be pathogenic. A deletion at 2p16.3 disrupts NRXN1 and was present in an affected sibling. A de novo duplication at 15q13.1 spans APBA2. The proteins of these two genes interact directly and play a role in synaptic development and function. Both genes have been affected by CNVs in other neurodevelopmental disorders. Keywords: Array CGH We undertook a systematic search for CNVs in patients with schizophrenia using high resolution, whole genome tiling path BAC arrays. We selected 45 male and 48 female unrelated proband-parent trios from our sample of ~600 Bulgarian SZ trios recruited as described previously. In all cases IQ was > 70. The mean age of probands was 33.8 years (SD = 10.1, range 13-57 years). The mean age at onset of psychotic symptoms was 22.1 years (SD = 6.6, range 11-44 years). Sonicated patient and reference DNA was labelled by random priming (Bioprime Array CGH, Invitrogen, Carlsbad, CA) with Cy3 and Cy5 (Amersham Biosciences, Piscataway, NJ), respectively, and hybridized onto a tiling path BAC array, consisting of ~36,000 BAC clones obtained from several sources as described on our website (http://www.molgen.mpg.de/~abt_rop/molecular_cytogenetics/). All protocols are also provided on that website. For the analysis and visualization of array CGH data, our software-package CGHPRO was employed. For the assessment of copy number gains and losses, we used conservative log2 ratio thresholds of 0.3 and -0.3, respectively. Deviant signal intensity ratios involving three or more neighboring BAC clones were considered to be potentially pathogenic, unless they were covered by more than one known DNA copy number variant, as listed in the Database of Genomic Variants. No dye swap was done. Array CGH analysis was successful in 91 cases.
2008-12-24 | E-GEOD-8606 | biostudies-arrayexpress
Project description:Background All animals must be able to adapt to changes in nutrient quality and supply. One striking aspect of this adaptive response is the extension of lifespan when caloric intake is reduced. These animals are also more resistant to various stresses, including starvation. We have characterized the response of adult Drosophila males and females to starvation and sugar conditions using high density microarrays. Results We have organized the regulated genes into specific metabolic and physiologic pathways. Most of the highly regulated genes have a strong sex bias. In addition to expected sex specific changes, such as egg production in females, we found unexpected biases in sensory systems, amino acid and purine metabolism, and signaling pathways. Comparison of starvation to caloric restriction revealed numerous overlaps. Conclusions Our results support the notion that starvation response shares similarities to caloric restriction. They have relevance for sex specific differences in longevity and in response to nutrient conditions which alter lifespan. Nutrient Conditions and Fly handling Larvae were kept on apple juice agar plates with yeast, and the larvae were raised at 25°C. For the developmental control larvae were collected 39-41 h AEL, for the developmental time-points at 51-53 hours and every 12 hours until 99-101 hours. Adult flies were kept at 25°C in 50 ml PS-tubes flat bottom (Greiner bio-one, Frickenhausen, Germany) on standard fly food (16.5 g yeast, 81,5 g cornmeal, 8 g agar, 100 ml sugar beet sirup and 200 ml 10% Nipagin in EtOH). Flies were collected that had hatched in 24 hours and kept in groups of ~200 flies/tube. After 5 days we separated males and females and put them in groups of 80/28 ml PS-tube. We allowed for one day CO2 recovery on standard fly food before flies were moved to 1) tubes with yeast paste on PBS soaked filter paper (control) 2) tubes with only PBS soaked filter paper (starvation) or 3) tubes with filter paper soaked in PBS with 20% Sucrose (sugar). After 24 or 48 hours flies were shock frozen in liquid nitrogen and stored at - 80°C for further handling. Males and females were assayed separately. For females, we performed 24 and 48 starvation, and 24 and 48 hours sugar. For the males only 24 hour starvation was done, since most died upon 48 hour starvation; and 24 and 48 hour sugar. Briefly, flies were collected within a 24 hour period, allowed to stay together for 4 days, separated into males and females, and allowed one day recovery in normal fly food. Afterwards, the flies were placed into three conditions: fresh yeast plus PBS, PBS or PBS plus 20 % sucrose. These experiments essentially parallel the experimental set up of previous larval experiments (7). The starvation and PBS were then compared with yeast (control).
2010-06-05 | E-GEOD-1672 | biostudies-arrayexpress
Project description:Mice used in this study were 129/sv males, 8-15 weeks of age, which were housed individually and received water ad libitum. Normal fed animals were fed before and during the 48h experiment with standard food ad libitum. Sugar fed animals were fed before the experiment with standard food ad libitum and during the 48h experiment with 50% (w/v) sugar solution (40% glucose, 10% sucrose). Mice were killed by CO2 asphyxiation. The livers were snap-frozen in liquid nitrogen and stored at -80 degrees C. Total RNA was prepared using the Nucleospin RNA L kit (Macherey-Nagel, Düren, Germany), with an additional step in which the final eluate was used for a subsequent elution step. PolyA RNA was extracted with the Ambion (Ambion Inc, Austin, USA) Purist kit according to the manufacturer's protocols. Equal amounts of polyA RNA were pooled to generate pools of all animals. Pool1, normal = 3 animals (N1, N2, N3), pool2, normal = 3 animals (N4, N5, N6), pool3, = 4 animals (N7, N8, N9, N10), pool1, sugar = 3 animals (Su1, Su2, Su3), pool2, sugar = 3 animals (Su4, Su5, Su6), pool3, sugar = 4 animals (Su7, Su8, Su9, Su10). Labeled cDNA was synthesized from 1.5 µg polyA RNA using the Amersham (Amersham Europe, Freiburg, Germany) direct cDNA labeling kit. Removal of the unincorporated dyes and concentration of the target were done with Microcon 30 (Millipore, Bedford, MA) spin columns according to the manufacturer's instructions. The concentrated probes were hybridized to the microarray in 1x dig easy hyb buffer (Hoffman-la Roche, Basel, CH) overnight at 42 degrees C. After hybridization the microarrays are washed 5’ in 2x SSC, 0.1% SDS at 42 degrees C; 10’ in 0.1x SSC, 0.1% SDS at RT; 4 times 1’ in 0.1x SSC at RT and shortly dipped into 0.01x SSC. Drying is performed in centrifuge on a microtiterplate holder at 130 rcf for 7’. Slides are stored in dark until they are scanned. Arrays were scanned using the dual laser scanner Axon 4000B and the corresponding software Genepix 4 (Axon Inc., Union City, CA, USA). Both channels (532 nm for Cy3 and 635 nm Cy5) were scanned in parallel and stored as 16 bit tiff files. The absolute intensity values span the range from 0 to 65,535. The scans were performed with a resolution of 10 µm. From each spot with a mean diameter of 100 µm, 100 data pixels were recorded. Individual local background area around the spots were defined, which included approximately 400 pixels and excludes neighboring spots. For each channel, the raw data was calculated as the median intensity of all foreground pixels with respect to all background pixels. Each array was scanned three times (low, medium and high scan) with different signal amplification factors (voltage settings of the photo multiplier tubes), but with the same laser power. The channels for Cy3 and Cy5 were balanced in each scan for approximately the same intensity profile. In the low scan no spot was saturated, in the high scan the signal amplification for Cy5 was set to approximately 80% of maximum and the Cy3 amplification was adjusted to this. The settings used in the medium scan lie between the low and the high scan. This method for scanning has several advantages. In the low scan where no spot is in saturation, it is possible to calculate the real ratio for genes with high expression levels; however, those with a low expression level are most likely not recognized. In order not to lose these a high scan is made; in this case the information on the saturated spots is lost so the two scans complement each other. The medium scan produces additional values for subsequent calculations. By scanning the arrays three times errors which occur while recording and which might increase the error factor in the normalization are averaged. The data processing was automated in a Visual Basic/Excel (Microsoft) program, which integrates the following steps. Raw data are derived from the result files generated by scanning and picture analysis software Genepix. The spot diameter is to lie in the range from 80 to 120 µm otherwise these spots are not included in analysis. From the pixel to pixel ratios between the foreground values of both colors, the standard deviation (SD) is calculated. Those spots which show a ratio SD of higher than 3 are excluded from analysis due to inconsistency. The foreground signal of each spot is corrected by subtracting the corresponding local background. Resulting values which are lower than the background are replaced by the background value. This defines the minimum measurement threshold as the background and avoids calculations of completely wrong ratios, e.g., if the background corrected value for one channel is close to zero. Spots are marked as saturated in one channel if more than 10% of the foreground pixels are at the highest absolute value. From the unsaturated data points of all three scans, linear regression between the low and medium, and between low and high scans, are calculated for both colors. Taking these regressions, the values of saturated spots are replaced by estimated ones which reflect the real intensity. The generated data set of each scan is normalized afterwards based on the intensity-dependent methods as described by Yang et al (Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J, and Speed TP. Normalization for cDNA microarray data: robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 30: e15, 2002). The ratios are calculated as log2 transformed values: M=log2 (Cy5/Cy3). Number describing the spot intensity A=log2sqrt (Cy5*Cy3) is calculated according to Yang et al. A within pin-tip group loess fit to the MA-plot was made. The loess scatter plot smoother performs robust locally fits using a tricube function to weight the values relative to the median point in the intensity interval. As interval size 20% was chosen, which corresponds to 180 data points in a pin-tip group containing 30 by 30 spots. After combining the normalized data from all blocks the data sets of all scans are additionally normalized using a global approach. The normalization was performed so that the sum of all log transformed ratios (M) is 0. All statistical normalizations are based on the assumption that (1) the majority of genes are not changed in their expression and (2) that the overall up and down regulations statistically compensate each other in sum. Taking this into account, the standard deviation in the ratios of the stable genes (80% of the most unchanged genes) could be seen as the measurement noise of the array experiments. To be able to compare the different scans and different normalized microarrays, the SD of the stable genes ratios was adjusted to a medium estimated value of 0.2, which is dependent on the used array system. This value does not influence the subsequent statistical analysis because the normal distribution of the data is not changed. For each microarray the normalized and adjusted log ratios of the three scans are averaged. Keywords = microarray analysis Keywords = nutrient response Keywords = caloric restriction Keywords = metabolic signaling Keywords = aging/longevity This SuperSeries is composed of the following subset Series: GSE851: liver, 48h sugar GSE852: liver, 48h starved GSE853: liver, 24h starved Refer to individual Series
2004-02-08 | E-GEOD-858 | biostudies-arrayexpress
Project description:Nasopharyngeal carcinoma (NPC) remains a majoy health problem worldwide, specially in Southeast China. In order to find the new candidate genes and molecular markers that are associated with nasopharyngeal carcinoma (NPC), this study focused on the screening NPC relative genes by gene expression profile. Keywords: disease state analysis 23 NPC biopsies and 15 nasopharynx chronic phlogistic biopsies were used to screen NPC relative genes by BioStarH-141s (2004) profile gene chips which contained 14112 points of full length human genes. The tumor samples were labeled with Cy5-dUTP.The nasopharyngeal phlogistic tissues were labeled with Cy3-dUTP. Biostatistics and bioinformatics were also used to analyse the differently expressed genes.
2009-04-21 | E-GEOD-11219 | biostudies-arrayexpress
Project description:Hepatocellular carcinoma (HCC) remains a major health problem worldwide, and HCC patients have a poor prognostic outcome. In this study, we systematically disclose mechanisms of hepatocarcinogenesis, and also effectively identify and validate novel anticancer targets in HCCs. Keywords: disease state analysis total 58 cDNA microarrays, all experiment samples are hepatocellular carcinoma. RNAs from 33 corresponding noncancerous livers and 5 normal livers were used as the reference, respectively. The tumor samples were labeled with Cy5-dUTP.The nontumor samples were labeled with Cy3-dUTP.
2009-01-06 | E-GEOD-4108 | biostudies-arrayexpress
Project description:Background Insulin signaling pathway is conserved from worms to humans. In Drosophila, three insulin-like peptides (dilps) are expressed in the insulin producing cells (IPCs) in the brain. In order to identify target genes of insulin signaling, the IPCs of female flies were destroyed during development by expressing apoptosis inducing factors reaper and head involution defective under control of the promoter region of dilp3. These insulin-deficient flies were analysed using high density microarrays. Results From the genes that appeared to be regulated in the absence of IPCs, the strongest difference was found for a gene involved in carbohydrate metabolism. Sequence analysis revealed that it is an alpha-glucosidase, which is able to break down disaccharides and/or glycogen. Conclusions We could identify an alpha-glucosidase, involved in carbohydrate metabolism, to be strongly affected in the context of decreased insulin signaling. The striking feature of this regulation was not only that it was the highest regulated, but it was the only gene regulated to this degree. Nutrient Conditions and Fly handling Experimental and control female flies were collected after hatching, then kept on fly maintenance food and separated from males after 11 days. After one day of recovery from carbon dioxide treatment, females were put in cages with PBS agar plates and supplied with yeast paste as only food source for 48 hours. Flies were shock frozen in liquid nitrogen and stored at - 80°C for further handling. Keywords: IPC knockout, 14 day old female flies, 48h yeast feeding Insulin producing cell deficient flies were compared to control flies. Two independent biological repeats were performed. From the two repeats, 5 chips were hybridised, including dye swap Experimental and control female flies were collected after hatching, then kept on fly maintenance food and separated from males after 11 days. After one day of recovery from carbon dioxide treatment, females were put in cages with PBS agar plates and supplied with yeast paste as only food source for 48 hours. The microarray was scanned 3 times: low scan (suffix _L in data table) with a low amplification / intensity to avoid saturated spots for proper data analysis, high scan (suffix _H in data table) with a high amplification / intensity to detect also weak fluorescent spots which are missed in the low scan and medium scan which lies between high and low intensity and gives additional data points for subsequent calculations. Both copies and all hybridisation repeats were used for normalisation, VALUE therefore has the same value for these copies in all relevant hybridisations. For additional information see publication and web link. Keywords = insulin, aging, longevity, nutrient dependance Lot batch = FP7
2010-06-25 | E-GEOD-10625 | biostudies-arrayexpress
Project description:Prostate cancer is one of the most commonly diagnosed cancers among men in the world and the pathogenesis of prostate cancer remains poorly understood. In this study, we intended to disclose genes that might be involved in tumorigenesis using cDNA microarray technology. Keywords: disease state analysis A total of 28 experiments were performed without replicates, using a pool of unrelated fetal tissues as common references. 25 experiments were done for prostate cancer versus common reference and 3 for normal prostate versus common reference. Prostate cancer and normal prostate samples were labeled with Cy5-dUTP. Common references were labeled with Cy3-dUTP.
2008-09-25 | E-GEOD-5957 | biostudies-arrayexpress
Project description:Lobular intraepithelial neoplasia grade 3 (LIN) is a recently recognized variant of intraepithelial lobular neoplasia of the breast composed of either uniform, generally small, cells (classic) with massive lobular distension, pleomorphic cells, signet-ring cells, or any cell type with necrosis. In contrast to classic forms of LIN, there is no consensus on therapeutic strategies for LIN3. In part this is due to the paucity of molecular data that could assist in defining the relationship of LIN3 to classic LIN and carcinomas. In this study we have employed array CGH to determine the patterns of chromosomal aberrations in 9 LIN3 lesions. By comparison to array CGH data of 13 classic LIN lesions, we demonstrate that LIN and LIN3 share several recurrent changes, in particular gains of 1q and losses of 16q. Both aberrations are known to appear early in tumorigenesis and to be associated with good prognosis. However, apart from this overlap, there were a number of karyotypic features that were observed exclusively in LIN3. Clearly, this lesion was characterized by a significantly higher number of DNA copy number changes (9 vs. 31 on average), a considerable complexity of chromosomal rearrangements with up to 16 breakpoints in one chromosome and overlapping high copy amplifications. The amplicons encompassed a number of known oncogenes such as ERBB2, CyclinD1 and FGF3 that have already been reported to be overexpressed in breast carcinomas. Our data suggest that, at the genetic level, LIN3 represents a highly advanced lesion with considerable resemblance to carcinomas and, therefore, reflects a lesion on the verge of invasion, and might represent the transition state from an intraepithelial neoplasm to breast carcinoma. In this study, nine cases of pleomorphic/necrotic LIN lesions as well as one classic LIN and the concurrent invasive lobular carcinoma (ILC) were analyzed by array CGH. Sections of formalin-fixed paraffin-embedded (FFPE) specimens cut at 7-8µm were mounted on special foil-coated slides (Molecular Devices, San Diego, USA). The sections were then deparaffinized with Xylene, processed in decreasing concentrations of ethanol and stained with haematoxylin. Lasercapture microdissection for both lesions was performed at multiple sites using Veritas Arcturus. For reference-DNA, female mammary tissue without histomorphological changes obtained from reduction mammoplasty specimens was procecessed and laser-microdissected as explained above. Cells were digested in 10µl TE, pH 9, and 0.5µl proteinase K (20mg/ml) for 48h at 55°C. After inactivation of proteinase K at 99°C for 10min, the digest was stored at -20°C. Without any further purification, the complete digest was used for whole genome amplification by means of the WGA (Whole Genome Amplification) kit from Sigma following the manufacturerâs recommendations. Array CGH was performed as described previously. In brief, two µg of amplified tumor and reference DNA were labelled by random priming (BioPrime® Total Genomic Labeling System, Invitrogen, Carlsbad, CA) with Alexa Fluor® 3 and Alexa Fluor® 5, respectively, and hybridized onto a tiling path BAC array, consisting of the human 32k BAC Re-Array Set (BACPAC Resources Center; http://bacpac.chori.org/pHumanMinSet.htm; DNA kindly provided by Pieter de Jong) and a 1Mb Resolution BAC set (clones kindly provided by Nigel Carter, Wellcome Trust Sanger Centre). All protocols are provided in detail on our website (http://www.molgen.mpg.de/~abt_rop/molecular_cytogenetics/) and more information concerning this platform have been submitted to the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/; GLP: 5114). For the analysis and visualization of array CGH data, our software-package CGH-PRO was employed. No background subtraction was applied. Raw data were normalized by âSubgrid LOWESSâ. For the assessment of copy number gains and losses, we used circular binary segmentation in combination with log2 ratio thresholds of 0.15 and -0.15, respectively. High copy amplifications were defined by focal appearance and a log2 ratio exceeding 0.45. In order to statistically investigate the similarity of aberration profiles we applied Pearson's product-moment correlation to cbs ratios of each of the corresponding arrays.
2010-06-20 | E-GEOD-14803 | biostudies-arrayexpress