Project description:Mammalian cells were grown as multicellular aggregates (spheroids) in an effort to determine the signaling events required for two cellular transformations states; primary foreskin fibroblasts (HFF-2) and glioblastoma cancer (T98G) cells, to survive at room temperature under oxygen and nutrient-deprived conditions for extended periods of time (2 weeks) and subsequently grown out from the arrested state as adherent monolayers. HFF-2 cells were cultured in DMEM supplemented with 15% fetal bovine serum and 5% carbon dioxide humidified air at 37 degrees C. T98G cells were cultured in EMEM with 10% FBS, 5% non-essential amino acids and 5% carbon dioxide humidified air at 37 degreesC. Monolayers were grown in T-185 flasks to 60% confluency then split into T-185 flasks coated with a 1% agarose mix in a 2:1 media/water ratio. Cells were suspended in 30 ml of supplemented media and grown for 4 days in order to form multicellular spheroids as described previously by our group (J. Cell. Physiol., 206 [2006] 526-536; see GSE1364 and GSE1455 for similar experiments with HEK293 cells). The suspension was removed from the flasks and centrifuged (1500 x g, 2 min) and the media removed. The pellet was returned to the flasks and then placed in vacuum bags (Dri-shield 2000 moisture barrier bag from Surmount Inc., USA; Cat. number 70068), which were sealed immediately under vacuum (Deni Magic Vac, Champion model; Keystone Manufacturing, USA). Vacuum-sealed flasks were stored for 2 weeks (in the dark) at room temperature. Recovery was initiated by removing the flask from the bag and resuspending the spheroids in supplemented media and placing the flasks in a 5% CO2/humidified air incubator maintained at 37 degreesC. Timepoints for transcriptional analysis were monolayer (control), 4 day growth spheroids, 2 week stored spheroids and 7 day growth back to monolayers. There were 67 HFF-2 and 50 T98G genes that exhibited at least a 10-fold change in expression during the course of arrest and recovery. Eleven of these genes were common to both cell lines. While the trends were the same between the two cell lines for genes that were in common, the timing of the responses were clearly different. The primary fibroblasts showed significant gene expression changes during spheroid formation and returned to their initial monolayer state after the two week arrest, whereas the glioblastoma cells exhibited major changes in gene expression during the recovery process. Furthermore, the T98G cells never returned to the monolayer expression levels during the time course of the recovery phase (7 days growth under monolayer conditions). Major differences in the transcriptional data were also found between the cell lines with regards to the putative cellular location of the encoded proteins. Of the genes with significant fold changes (+/- 10-fold) for which location information is available, the primary fibroblasts were significantly enriched in genes encoding extracellular (32%) and membrane-associated (26%) proteins. The glioblastoma cells, on the other hand, had only 17% extracellular and 13% membrane-associated. The expression level trends for all genes above the 10-fold cutoff were as observed for the "in common" genes; HFF-2 genes had returned to monolayer expression levels after 7 days of recovery whereas the T98G line was still exhibiting expression values much different from that of the monolayer control.

Project description:Using a chromatin immunoprecipitation-paired end diTag cloning and sequencing strategy, we mapped estrogen receptor alpha (ERalpha) binding sites in MCF-7 breast cancer cells. We identified 1,234 high confidence binding clusters of which 94% are projected to be bona fide ERalpha binding regions. Only 5% of the mapped estrogen receptor binding sites are located within 5 kb upstream of the transcriptional start sites of adjacent genes, regions containing the proximal promoters, whereas vast majority of the sites are mapped to intronic or distal locations (>5 kb from 5' and 3' ends of adjacent transcript), suggesting transcriptional regulatory mechanisms over significant physical distances. Of all the identified sites, 71% harbored putative full estrogen response elements (EREs), 25% bore ERE half sites, and only 4% had no recognizable ERE sequences. Genes in the vicinity of ERalpha binding sites were enriched for regulation by estradiol in MCF-7 cells, and their expression profiles in patient samples segregate ERalpha-positive from ERalpha-negative breast tumors. The expression dynamics of the genes adjacent to ERalpha binding sites suggest a direct induction of gene expression through binding to ERE-like sequences, whereas transcriptional repression by ERalpha appears to be through indirect mechanisms. Our analysis also indicates a number of candidate transcription factor binding sites adjacent to occupied EREs at frequencies much greater than by chance, including the previously reported FOXA1 sites, and demonstrate the potential involvement of one such putative adjacent factor, Sp1, in the global regulation of ERalpha target genes. Unexpectedly, we found that only 22%-24% of the bona fide human ERalpha binding sites were overlapping conserved regions in whole genome vertebrate alignments, which suggest limited conservation of functional binding sites. Taken together, this genome-scale analysis suggests complex but definable rules governing ERalpha binding and gene regulation. Experiment Overall Design: We used oligonucleotide expression microarrays (Affymetrix GeneChip U133 Plus 2.0) to identify estradiol (E2)-responsive genes in the estrogen-receptor positive breast cancer cell line, MCF7. MCF7 cells were grown to 30-50% confluency and exposed to 10 nM E2 (or vehicle only) at 12, 24, and 48 hours. Each timepoint was performed in triplicate (ie, biological replicates). Total RNA was isolated from cells using the Qiagen RNeasy kit, and 5 micrograms of total RNA was amplified, labeled and hybridized to the array according to the manufacturer’s protocols.

Project description:BackgroundPrednisone, one of the most highly prescribed drugs, has well characterized effects on gene transcription mediated by the glucocorticoid receptor. These effects are typically occurring on the scale of hours. Prednisone also has a number of non-transcriptional effects (occurring on minutes scale) on protein signaling, yet these are less well studied. We sought to expand the understanding of acute effects of prednisone action on cell signaling using a combination of SILAC strategy and subcellular fractionations from C2C12 myotubes.ResultsDe novo translation of proteins was inhibited in both SILAC labeled and unlabeled C2C12 myotubes. Unlabeled cells were exposed to prednisone while SILAC labeled cells remained untreated. After 0, 5, 15, and 30 minutes of prednisone exposure, labeled and unlabeled cells were mixed at 1:1 ratios and fractionated into cytosolic and nuclear fractions. A total of 534 proteins in the cytosol and 626 proteins in the nucleus were identified and quantitated, using 3 or more peptides per protein with peptide based probability < or = 0.001. We identified significant increases (1.7- to 3.1- fold) in cytoplasmic abundance of 11 ribosomal proteins within 5 minutes of exposure, all of which returned to baseline by 30 min. We hypothesized that these drug-induced acute changes in the subcellular localization of the cell's protein translational machinery could lead to altered translation of quiescent RNAs. To test this, de novo protein synthesis was assayed after 15 minutes of drug exposure. Quantitative fluorography identified 16 2D gel spots showing rapid changes in translation; five of these were identified by MS/MS (pyruvate kinase, annexin A6 isoform A and isoform B, nasopharyngeal epithelium specific protein 1, and isoform 2 of Replication factor C subunit 1), and all showed the 5' terminal oligopyrimidine motifs associated with mRNA sequestration to and from inactive mRNA pools.ConclusionWe describe novel approaches of subcellular proteomic profiling and assessment of acute changes on a minute-based time scale. These data expand the current knowledge of acute, non-transcriptional activities of glucocorticoids, including changes in protein subcellular localization, altered translation of quiescent RNA pools, and PKC-mediated cytoskeleton remodeling.

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Transcription profiling of camptothecin treatment time series performed on human DBTRG-05MG glioma cells

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