Expression microarray analysis of acquired gemcitabine resistance
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Paul van den IJssel
PROVIDER: E-GEOD-2352 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
Expression microarray analysis and oligo array comparative genomic hybridization of acquired gemcitabine resistance in mouse colon reveals selection for chromosomal aberrations.
van de Wiel Mark A MA Costa Jose L JL Smid Kees K Oudejans Cees B M CB Bergman Andries M AM Meijer Gerrit A GA Peters Godefridus J GJ Ylstra Bauke B
Cancer research 20051101 22
Gemcitabine is a commonly used therapy for many solid tumors. Acquired resistance to this nucleoside analogue, however, diminishes the long-term effectiveness in a majority of patients. To better define the molecular background of gemcitabine resistance, a mouse colon tumor was selected during successive rounds of transplantation with continued treatment of gemcitabine. Expression microarray analysis was applied to determine which genes are consistently and highly overexpressed or underexpressed ...[more]
Similar Datasets
Project description:Hybridization of one kidney of cortisol treated fish vs. one kidney of control fish. Kidneys were collected from untreated juvenile sea bream (n=4) and from fish, which received for 72h a coconut-oil implant containing 10mg/Kg (fish wet weight) (n=4) cortisol. Experiments were carried out at the University of the Algarve, Portugal in accordance with National legislation for the welfare of animals. Experiments were conducted in two 125 l cylindriconical tanks supplied with a continuous through-flow of oxygenated seawater at 20+1 °C using juvenile sea bream (25 g + 3 g) adapted for 1 week to the experimental conditions. One tank contained 8 untreated fish (control) and the other tank 8 cortisol treated fish and the end of experiments fish were removed form tanks, decapitated and the kidneys rapidly removed and place in RNAlater (Qiagen) at â20 °C. No mortality occurred in the control tank but 2 fish died in the cortisol treated tank.
2010-06-11 | E-GEOD-2064 | biostudies-arrayexpress
Project description:Five different developmental stages, neurula (1), hatching (48 h post-fertilization) (2), pectoral fin budding (96 h post-fertilization) (4), mouth opening (144 h post-fertilization) (6), and eye pigmentation (168 h post-fertilization) (7) were compared directly to each other in all possible combinations (Loop design).All experiments were perfomed by dye swap hybridizations.
2010-06-11 | E-GEOD-1887 | biostudies-arrayexpress
Project description:Drosophila melanogaster adult flies fed on food containing 16 mg/ml of pentylenetetrazole (PTZ) in the food show a hyperkinetic behavior within 24 hours. Half of that concentration, i.e., 8 mg/ml, of PTZ, if fed for seven days, though doesn’t cause seizure-like behavior, results in a decreased climbing speed in flies. This change in locomotor behavior is progressive and becomes significant only on seventh day of the treatment. We also examined flies’ locomotor behavior secondary to PTZ withdrawal. Interestingly, an increased climbing speed was found to develop seven days after withdrawal. Importantly, antiepileptic drugs showed effectiveness in the above fly model. Earlier, we submitted in GEO time series of fly head microarray gene expression profiling during chronic PTZ and PTZ withdrawal phase. We also submitted previously expression profiles associated with antiepileptic drug treatment. Here, we have undertaken a different line of work. Having developed a well characterized acquired model of behavioral and gene expression plasticity, we found an opportunity here to investigate if drug exposure to adult males could cause transgenerational effect. To probe this, we carried out a systematic study at both behavioral and microarray gene expression levels. In the latter, we asked the question that do F0 testis, F1 males’ head, F1 females’ head, F1 testis, F2 males’ head and F2 females’ head show gene expression changes if F0 male parents had a history of PTZ exposure? A total of 28 microarray slides were used in this study. Unless mentioned otherwise, standard method of fly handling and manipulation was used. Freshly generated isogenic D. melanogaster Oregon-R wild type flies were grown in standard fly medium consisting of agar-agar, maize powder, brown sugar, dried yeast, and nipagin. The cultures were grown at 24 + 1oC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Three to four days old unmated adult males were grown in food containing 8 mg/ml PTZ for seven days. Flies were then shifted to vials containing NF. Seven days after shifting, F0 males were mated to same age group females maintained on NF throughout, without any drug exposure. Males and females were housed in groups in vials containing NF. Eggs collected on NF were used to generate F1 individuals as unmated males and virgin females. F1 males were mated to same age group females which were independently collected and maintained on NF. These females had no history of drug exposure in prior generations. Males and females were housed in groups in vials containing NF. Eggs collected on NF were used to generate F2 individuals as unmated males and virgin females. F0 testis, F1 males’ head, F1 females’ head, F1 testis, F2 males’ head and F2 females’ head were used for RNA isolation. Total RNA was isolated from pools of heads or testis, using TRI REAGENT (Sigma) according to the manufacturer’s protocol. Double stranded cDNA was synthesized from extracted RNA using Microarray cDNA Synthesis Kit (Roche). The cDNA was purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. Each of the four sets of control (no PTZ exposure of F0 males) and treated cDNA samples, belonging to the four biological replicates, was used for labeling with either Cy3 or Cy5 dyes (Amersham Biosciences) using Microarray RNA Target Synthesis Kit T7 (Roche). The labeled products were purified by Microarray Target Purification Kit (Roche). The Cy3 and Cy5 labeled two cRNA samples of each biological replicate were pooled together, precipitated, washed, air-dried, and dissolved in 18MΩ RNAase free water (Sigma). Dye swapping was accomplished by hybridizing two arrays with control as Cy3- and experimental as Cy5- labeled sample, and the rest two as the opposite, control as Cy5- and experimental as Cy3- labeled sample. Two sets of microarrays were generated for F1 males. The labeled product was mixed with hybridization solution containing hybridization buffer (DIG Easy Hyb; Roche), 10 mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10 mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma). The hybridization mixture was denatured at 65ºC and applied onto cDNA microarray slides (D14Kv1, CDMC, Toronto, Canada). The slides were covered by a coverslip (ESCO, Portsmouth, USA) and hybridization was allowed to take place in hybridization chamber (Corning) at 37ºC for 16 hrs. Following hybridization, the coverslips were removed in a solution containing 1X SSC and 0.1% SDS at 50ºC, and the slides washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50ºC with occasional swirling and then transferred to 1X SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Slides were given a final wash in 0.1X SSC for 15 minutes and then liquid was quickly removed from the slide surface by spinning at 600 rpm for 5 minutes. Slides were scanned at 10 µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices). The preprocessing and quantification of the 16 bit TIFF images were carried out using Gene Pix Pro 6.0 software (Molecular Devices). Ratio based normalization was performed using Acuity 4.0 software (Molecular Devices). All Spots with raw intensity less then 100U and less then twice the average background was ignored during normalization. Normalized data was filtered for the selection of features before further analysis. Only those spot were selected which contained only a small percentage (<3) of saturated pixels, were not flagged bad or found absent (flags >= 0), had relatively uniform intensity and uniform background (Rgn R2 (635/532) >= 0.6) and were detectable above background (SNR >= 3). Analyzable spots in at least three of the four biological replicates performed were retrieved for downstream analysis using Significance Analysis of Microarrays (SAM 3, Excel Add-In, Stanford) under the conditions of one class response, imputation and 100 permutations. Analysis was also carried out using fold-change criterion in Acuity 4.0 itself.
2009-03-25 | E-GEOD-15136 | biostudies-arrayexpress
Project description:Ferric uptake regulator (Fur) is the major regulator of iron acquisition in Escherichia coli and other bacteria. In the present study, the role of Fur in anaerobic S. enterica serovar Typhimurium was determined by transcriptome analysis, reporter assays, and enzymatic assays. In anaerobic ?fur, 298 genes were differentially expressed. In general, Fur repressed genes required for iron acquisition/storage, metabolism, electron transport, oxidative/nitrosative stress, and modulators of virulence. There were 73 genes whose expression required Fur, eleven of which contain a putative Fur box 5? of the gene. Transcription of sodA was >9-fold higher in ?fur but there was no corresponding increase in the activity of SodA. Anaerobic cultures of the fur mutant and the WT were used to inoculate two sets of three independent flasks each containing 150 ml of anoxic LB-MOPS-X. The three independent cultures were grown to an optical density at 600 nm (OD600) of 0.25 to 0.35, pooled, and treated with RNAlater (QIAGEN, Valencia, CA) to fix the cells and preserve the quality of the RNA. Total RNA was extracted with the RNeasy RNA extraction kit (QIAGEN), and the samples were treated with RNase-free DNase (Invitrogen, Carlsbad, CA). The absence of contaminating DNA and the quality of the RNA was confirmed by PCR amplification of known genes and by using agarose gel electrophoresis. Aliquots of the RNA samples were kept at –80°C for use in the microarray and quantitative real-time reverse transcription-PCR (qRT-PCR) studies. The SuperScript Indirect cDNA labeling system (Invitrogen) was used to synthesize the cDNA for the hybridizations. Each experiment consisted of two hybridizations, on two slides, and was carried out in Corning Hybridization Chambers at 42°C overnight. Dye swapping was performed to avoid dye-associated effects on cDNA synthesis. The slides were washed at increasing stringencies (2x SSC [1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 0.1% sodium dodecyl sulfate [SDS], 42°C; 0.1% SSC, 0.1% SDS, room temperature; 0.1% SSC, room temperature). Following hybridization, the microarrays were scanned for the Cy3 and Cy5 fluorescent signals with a ScanArray 4000 microarray scanner from GSI Lumonics (Watertown, MA). The intensity of every spot was codified as the sum of the intensities of all the pixels within a circle positioned over the spot itself and the background as the sum of the intensities of an identical number of pixels in the immediate surroundings of the circled spot. Cy3 and Cy5 values for each spot were normalized over the total intensity for each dye to account for differences in total intensity between the two scanned images. The consistency of the data obtained from the microarray analysis was evaluated by a pair-wise comparison, calculated with a two-tailed Student's t test and analyzed by the MEAN and TTEST procedures of SAS-STAT statistical software (SAS Institute, Cary, NC) . The signal intensity at each spot from the fur mutant and the WT were background subtracted, normalized, and used to calculate the ratio of gene expression between the two strains. All replicas were combined, and the median expression ratios and standard deviations were calculated for open reading frames (ORFs) showing 2.5-fold change.
2011-10-13 | E-GEOD-18441 | biostudies-arrayexpress
Project description:We characterized the global response of plants carrying a mitochondrial dysfunction induced by the expression of the unedited form of the ATP synthase 9 subunit. The u-ATP9 transgene driven by A9 and Apetala3 promoters induce mitochondrial dysfunction revealed by a decrease in both oxygen uptake and ATP levels, with an increase in ROS and a concomitant oxidative stress response. The transcriptome analysis of young Arabidopsis flowers, validated by RT-PCR and enzymatic or functional tests, show dramatic changes in u-ATP9 plants. Both lines present a modification in the expression of several genes involved in carbon, lipid and cell wall metabolism, suggesting that an important metabolic readjustment occurs in plants with a mitochondrial dysfunction. Interestingly, transcript levels involved in mitochondrial biogenesis, protein synthesis, and degradation are affected. Moreover, several mRNA levels for transcription factors and DNA binding proteins were also changed. Some of them are involved in stress and hormone response, suggesting that several signaling pathways overlap. Indeed, the transcriptome data reveal that the mitochondrial dysfunction dramatically alters genes involved in signaling pathways, including those involved in ethylene, absicic acid and auxin signal transduction. Our data suggest that the mitochondrial dysfunction model used in this rapport may be useful to uncover the retrograde signaling mechanism between the nucleus and mitochondria in plant cells. Arabidopsis oligonucleotide microarrays fabricated by the University of Arizona contain 26,000 oligonucleotides (http://www.ag.arizona.edu/microarray/). RNA was isolated from 6-week-old flowers from u-ATP9 and wt plants. The experimental (mutant) and reference (wild type) RNA samples were reverse-transcribed and directly labeled with either Cy5-dUTP or Cy3-dUTP fluorescent dye (GE Healthcare), using random hexamer primers (Invitrogen). Excess nucleotides and primers were removed using QIAquick PCR Purification Kit (Qiagen). Labeled samples were mixed and then hybridized to microarray for 15 h at 60°C. The slides were washed at room temperature in three wash steps: 2 x SSC, 0.5% SDS; 0.5 x SSC; and 0.05 x SSC for 5 min each with gentle shaking. The slides were scanned with a GenePix 4000B Scanner (Axon Instruments Inc., Union City, CA). Normalization between the Cy3 and Cy5 fluorescent dye emission channels was achieved by adjusting the levels of both image intensities. The experiments were repeated four times with samples from different experiments, as biological replicates. In dye swapping experiments, the RNA samples from different experiments were reciprocally labeled, both as a biological and technical repetition for comparing the reproducibility of the experiments. Hybridization intensities for each microarray element were measured using ScanAlyze 4.24 (available at http://genome-www4.stanford.edu/MicroArray/SMD/restech.html). The two channels were normalized in log space using the z-score normalization on a 95% trimmed data set. We removed unreliable spots according to the following criteria: spots flagged as having false intensity caused by dust or background on the array were removed; and spots for which intensity was less than three fold above background were also eliminated. Data from multiple experiments were normalized (Bolstad, 2003) and signals from spots from different experiments were statistically analyzed using Significance Analysis of Microarrays using the one class response (SAM, http://www-stat.stanford.edu/~tibs/SAM/), cut at a false discovery rate < 10% (Tusher, 2001).
2009-02-15 | E-GEOD-14240 | biostudies-arrayexpress
Project description:A common bacterial strategy for monitoring environmental challenges is to use two-component systems, which consist of a sensor histidine kinase (HK) and a response regulator (RR). In the food-borne pathogen Bacillus cereus, the alternative sigma factor ÏB is activated by the RR RsbY. Here we present strong indications that the PP2C-type phosphatase RsbY receives its input from the multi-sensor hybrid kinase BC1008 (renamed RsbK). Genome analyses revealed that, across bacilli, rsbY and rsbK are located in a conserved gene cluster. A B. cereus rsbK deletion strain was shown to be incapable of inducing ÏB upon stress conditions and was impaired in its heat adaptive response. Comparison of the wild-type and rsbK mutant transcriptomes upon heat shock revealed that RsbK was primarily involved in the activation of the ÏB-mediated stress response. Truncation of the RsbK RR receiver domain demonstrated the importance of this domain for ÏB induction upon stress. The domain architecture of RsbK suggests that in the B. cereus group and in other bacilli, environmental and intracellular stress signalling routes are combined into one single protein. This strategy is markedly different from the ÏB activation pathway in other low-GC Grampositives. Two different comparisons were performed (both in duplo and Cy5-Cy3 dye-swapped): (1) B. cereus WT 30°C versus B. cereus WT 42°C (10 min heat shock) (2) B. cereus WT 42°C versus B. cereus FM1404 42°C Data from comparisons (2) were subsequently compared with transcriptome data obtained previously by Van Schaik et al., 2007
2010-06-24 | E-GEOD-14589 | biostudies-arrayexpress
Project description:Samples and RNA preparation: The series is composed of 4 hybridizations for analysis of differentially expressed genes in the liver of ciprofibrate dosed vs. control rats. Rats (200-250 g) were given (by gastric intubation) tap water (control group, n=5) or ciprofibrate (50 mg/kg body weight, once daily for 60 days, n=4). Liver total RNA extraction was performed first by ultracentrifugation on a cesium chloride cushion and then using TRIZOL Reagent (GIBCO BRL Life Technologies, New York, NY). Labeling and hybridization: One microgram of total RNA from the control pool (n=5), and from each ciprofibrate dosed rat was reverse transcribed and labeled with Cy3- and Cy5-attached dendrimer, respectively, using 3DNA Array Detection Submicro kit as described in the manufacturer's protocols (Genisphere, Montvale, NJ). The labeling reactions were also spiked with exogenous control RNAs (Stratagene) at expected Cy5/Cy3 ratios of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 0.5, 0.33, 0.25 and 0.125 for control spike 1, 2, 3, 4, 5, 6, 8, 9, 10 and 7, respectively. The Cy3 and Cy5 labeled samples (combined volume 5 microliter) were mixed with 30 microliter of the hybridization solution containing 0.25 M NaPO4, 1 mM EDTA, 4.5% SDS, 1 x SSC, 2 x Denhardt's solution, 0.25 microliter antifade reagent (Genisphere) and 0.2 microgram mouse COT-1 DNA (GIBCO BRL Life Technologies), added onto the array and covered with 22 x 50mm cover slip. The array was assembled into humidified hybridization chamber (Corning) and hybridized at 65oC for 18 h by submerging in a water bath. Post-hybridization washes were done once in 2 x SSC, 0.2% SDS at 55oC for 15 min, then in 2 x SSC at room temperature for 15 min, and finally with 0.2 x SSC at room temperature for 15 min. Scanning, image analysis and data processing: Arrays were scanned at 10 micrometer resolution with a confocal laser scanner constructed in-house in collaboration with NEMKO (Trondheim, Norway) according to a prototype developed at NHGRI (http://www.nhgri.gov/DIR/LCG/15K(HTML/). Microarray image analysis was done with MicroArray Suite software version 2.1 with globally normalization (Scanalytics, Inc., Fairfax VA). First, spots undetected (target intensity=1) in at least one array were excluded. Then, spots with signal intensity (averages of the 4 arrays) less than 300 in both channels were removed. The fluorescence intensity ratios (Cy5/Cy3) for genes measured from probes spotted in duplicate were averaged for each slide. Then, mean ratios of the 4 hybridizations were calculated for each gene, and differentially regulated genes were determined using 1.4 fold change cutoff values (1.4 for up-regulated or 0.7 for down-regulated genes).
2010-06-05 | E-GEOD-335 | biostudies-arrayexpress
Project description:Pharmaceuticals are pseudo persistent aquatic pollutants with unknown effects at environmentally relevant concentrations. Atlantic salmon (Salmo salar) were exposed to Acetaminophen: 54.77 ± 34.67; Atenolol: 11.08 ± 7.98 and Carbamazepine: 7.85 ± 0.13 µg•L-1 for 5 days. After Acetaminophen treatment, 19 proteins were differently expressed, of which 11 were significant with respect to the control group (eight up-regulated and three down-regulated). After Atenolol treatment, 7 differently expressed proteins were obtained in comparison with the control, of which 6 could be identified (four up-regulated and 2 down-regulated). Carbamazepine exposure resulted in 15 differently expressed proteins compared with the control, with 10 of them identified (seven up-regulated and three down-regulated). Out of these, 3 features were common between Acetaminophen and Carbamazepine and one between Carbamazepine and Atenolol. One feature was common across all treatments. Principal component analysis and heat map clustering showed a clear grouping of the variability due to the applied treatments. The obtained data suggest (1) that exposure to environmentally relevant concentrations of the pharmaceuticals alters the hepatic protein expression profile of the Atlantic salmon; and (2) the existence of treatment specific processes that may be useful for biomarker development.
2014-11-18 | PXD001354 | Pride
Project description:Polycyclic aromatic hydrocarbons are common pollutants in soil, have negative effects on soil ecosystems, and are potentially carcinogenic. The Springtail (Collembola) Folsomia candida is often used as an indicator species for soil toxicity. Here we report a toxicogenomic study that translates the ecological effects of the polycyclic aromatic hydrocarbon phenanthrene in soil to the early transcriptomic responses in Folsomia candida. Microarrays were used to examine two different exposure concentrations of phenanthrene, namely the EC10 (24.95 mg kg-1 soil) and EC50 (45.80 mg kg-1 soil) on reproduction of this springtail, which evoked 405 and 251 differentially expressed transcripts, respectively. Fifty transcripts were differential in response to either concentration. Many transcripts encoding xenobiotic detoxification and biotransformation enzymes (phases I, II, and III) were upregulated in response to either concentration. Furthermore, indications of general and oxidative stress were found in response to phenanthrene. Chitin metabolism appeared to be disrupted particularly at the low concentration, and protein translation appeared suppressed at the high concentration of phenanthrene; most likely in order to reallocate energy budgets for the detoxification process. Finally, an immune response was evoked especially in response to the high effect concentration, which was also described in a previous transcriptomic study using the same effect concentration (EC50) of cadmium. Our study provides new insights in the molecular mode of action of the important polluting class of polycyclic aromatic hydrocarbons in soil animals. Furthermore, we present a fast, sensitive, and specific soil toxicity test which enhances traditional tests and may help to improve current environmental risk assessments and monitoring of potentially polluted sites. Folsomia candida was exposed to phenanthrene spiked soil or untreated (reference/control) soil for 2 days. Two different concentrations of phenanthrene were used, 24.95 and 45.80 mg/kg soil which represent the EC10 and EC50 on reproduction, respectively. For each concentration treatment 4 biological replicates were used, replicate samples consisted of total RNA extracted from ~30 animals exposed in the same jar to either reference or phenanthrene spiked soil. Phenanthrene treated samples were always hybridized to reference samples in an evenly distributed dye-swap manner, which resulted in total in 8 hybridizations of 16 samples.
2010-05-18 | E-GEOD-14207 | biostudies-arrayexpress
Project description:A total of 20 KYSE human esophageal squamous cell carcinoma cell lines (KYSE-30, -140, -150, -170, -180, -200, -220, -350, -410, -450, -510, -520, -590, -770, -850, -890, -1170, -1190, -1250, and -2270) and a non-cancerous esophageal epithelial cell (HEEC-1)were kindly provided by Dr. Y. Shimada (Kyoto University, Kyoto, Japan). The KYSE cell lines were cultured in RPMI 1640 medium (Life Technologies, Inc., Grand Island, NY) containing 10% heat-inactivated fetal bovine serum (FBS; BioWhittaker, Verviers, Belgium) in a humidified atmosphere of 5% CO2 and maintained in continuous exponential growth by passage every 3 days. HEEC-1 cells were cultured in Keratinocyte SFM medium with growth supplement containing 2.5 mg EGF and 25 mg bovine pituitary extract in 500 mL liquid basal medium (GIBCO BRL, Rockville, MD) and expanded by passage twice in a week. The exponentially growing cultured cells (2 x 10^6) were collected after two-washings with PBS. Total RNA was prepared from the frozen cell pellets using QIAGEN RNeasy mini kit (QIAGEN, Inc., Valencia, CA), and mRNA was purified using MACS mRNA Isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the supplierâs protocols. RIKEN human 21K array containing 20,784 clones with positive and negative controls was used to analyze gene expression of 20 KYSE esophageal cancer cell lines using HEEC-1 as reference. The target DNAs used to construct human 21 K array were the glycerol stock of cDNA clones purchased from ResGen (Invitrogen Corporation, Carlsbad, CA)). One-microgram poly(A) RNAs from KYSE cells and the reference cells were labeled with Cy3-dCTP and Cy5-dCTP, respectively, by random-primed reverse transcription. Arrays were laser-scanned using ScanArray 5000TM confocal laser scanner (GSI Lumonics, Billerica, MA), and the images were analyzed using ScanAlyzeTM (Stanford University). All experiments were performed in duplicate (_1 & _2). To explore the genes responsible for chemosensitivity in cancer cells, expression profiles of various cancer cell lines were examined. Statistically comparing them with the chemosensitivity measured by in vitro MTT assay in each cell line, several candidate genes were selected and predicition models for anticancer efficacy were established using their expression levels.
2010-06-05 | E-GEOD-2454 | biostudies-arrayexpress