Transcription profiling by array of human glioblastoma cell lines
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ABSTRACT: Recent studies demonstrated that tumor cells with stem cell-like properties can be cultured from human glioblastomas by using conditions that select for the expansion of neural stem cells. We established glioblastoma stem-like (GS-) cell cultures from 9 different glioblastomas, 8 of which generated stably expandable cell lines. Analyzing GS-cell cultures, we discovered two clearly discernable phenotypes. Microarray analysis showed that the 4 GSf cell lines shared expression profiles dominated by genes involved in nervous system development and neuropeptide signaling, while the 5 GSr lines shared expression signatures enriched for extracellular matrix-proteins. Experiment Overall Design: Affymetrix HG-U133 Plus 2.0 chips were employed for expression profiling from 9 different glioblasoma derived neurosphere cultures in two biological replicates.
Project description:We compared a large panel of human glioblastoma stem-like (GS) cell lines, corresponding primary tumors and conventional glioma cell lines to identify cell lines that preserve the transcriptome of human glioblastomas most closely, thereby allowing identification of shared therapeutic targets. We used Affymetrix HG-U133 Plus 2.0 microarrays to compare human glioblastoma stem-like (GS) cell lines, corresponding primary tumors and conventional glioma cell lines. We extracted total RNA from 32 conventional glioma cell lines, 12 GS cell lines (8 in two different passages), 7 clonal sublines derived from two GS lines, 12 original tumors, and 4 monolayer cultures established from the same tumors as GS-lines using standard serum conditions.
Project description:"We have developed a nonheuristic genome topography scan (GTS) algorithm to characterize the patterns of genomic alterations in human glioblastoma (GBM), identifying frequent p18INK4C and p16INK4A codeletion. Functional reconstitution of p18INK4C in GBM cells null for both p16INK4A and p18INK4C resulted in impaired cell-cycle progression and tumorigenic potential. Conversely, RNAi-mediated depletion of p18INK4C in p16INK4A-deficient primary astrocytes or established GBM cells enhanced tumorigenicity in vitro and in vivo. Furthermore, acute suppression of p16INK4A in primary astrocytes induced a concomitant increase in p18INK4C. Together, these findings uncover a feedback regulatory circuit in the astrocytic lineage and demonstrate a bona fide tumor suppressor role for p18INK4C in human GBM wherein it functions cooperatively with other INK4 family members to constrain inappropriate proliferation. Experiment Overall Design: Expression profiles of human glioblastoma frozen tumors and cell lines were obtained to study copy number abberation driven expressin alteration. Experiment Overall Design: Affymetrix human genome U133plus2 arrays were used to obtain the expression profiles of human glioblastoma tumors and cell lines."
Project description:Glioblastoma, the most aggressive and least treatable form of malignant glioma, is the most common human brain tumor. Although many regions of allelic loss occur in glioblastomas, relatively few tumor suppressor genes have been found mutated at such loci. To address the possibility that epigenetic alterations are an alternative means of glioblastoma gene inactivation, we coupled pharmacological manipulation of methylation with gene profiling to identify potential methylation-regulated, tumor-related genes. Triplicates of three short-term cultured glioblastomas were exposed to 5?M 5-aza-dC for 96 hours followed by cRNA hybridization to an oligonucleotide microarray (Affymetrix U133A). We based candidate gene selection on bioinformatics, RT-PCR, bisulfite sequencing, methylation-specific PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Two genes identified in this manner, RUNX3 and Testin (TES), were subsequently shown to harbor frequent tumor-specific epigenetic alterations in primary glioblastomas. This overall approach therefore provides a powerful means to identify candidate tumor suppressor genes for subsequent evaluation and may lead to the identification of genes whose epigenetic dysregulation is integral to glioblastoma tumorigenesis. Duplicates of three short term cultured glioblastoma cell lines (internal IDs: GLI56;GLI60;GLI72) were either exposed to 5umol 5`aza-dC for 96h or left untreated. Total RNA was extracted of treated and untreated cells after 96h and hybridized to U133A chip. Gene expression profiles of treated and untreated cells were compared. Upregulation of gene expression in the treated (demethylated) samples was interpreted as potentially being regulated by methylation - pointing towards hypermethylation in the related cell line. Identification of novel tumor suppressor genes, regulated by methylation, was the overall goal.
Project description:Expression profiles of 17 melanoma cell lines were analysed to identify genes differentially expressed between cell lines harbouring wild-type or mutant p16INK4A. Relevant paper: Pavey et al. (2007). Note: all of these cell lines contained wild-type p14ARF, so that the transcriptional effects of p16INk4A could be determined without interference from p14ARF. Experiment Overall Design: The aim of this study was to identify genes which are transcriptional targets of p16INK4A in melanoma.
Project description:We used microarrays to analyze the global expression patterns for 22 commercially available pancreatic cancer cell lines 22 pancreatic cancer cell lines were grown to 80% confluence in DMEM/FBS/PenStrep media and then harvested for total RNA
Project description:Human Glioblastoma Multiforme tumors taken before dendritic cell vaccination, the recurrent tumors taken after vaccination and control GBM tumors from non vaccinated patients. Experiment Overall Design: Six Glioblastoma Multiforme patients underwent surgery. Their brain tumors were removed and analyzed via microarray. The lysate from the tumors were cultured with the patients' dendritic cells and the DCs were injected back into the patients. The patients GBMs returned and they underwent surgery a second time and those tumors were also analyzed via microarray. Tumors from the first and second GBM surgeries of 5 patients who did not receive DC vaccines are included as controls.
Project description:A significant fraction of breast cancers exhibit de novo or acquired resistance to estrogen deprivation. To model resistance to aromatase inhibitor (AI) therapy, long-term estrogen-deprived (LTED) derivatives of MCF-7 and HCC-1428 cells were generated through culture for 3 and 7 months under hormone-depleted conditions, respectively. These LTED cells showed sensitivity to the ER downregulator fulvestrant under hormone-depleted conditions, suggesting continued dependence upon ER signaling for hormone-independent growth. To evaluate the role of ER in hormone-independent growth, LTED cells were treated +/- 1 uM fulvestrant x 48 h before RNA was harvested for gene expression analysis. MCF-7/LTED and HCC-1428/LTED cells were treated with 10% DCC-FBS with or without the estrogen receptor antagonist drug fulvestrant for 48 hrs prior to RNA harvest for array analysis. Three replicates per condition.
Project description:To identify patterns of gene expression that correlate with response to treatment with either melphalan or temozolomide we measured both gene expression using microarray genechips and response to drug using a standard in vitro cell proliferation assay. Senstivity to melphalan was measured 48hrs after drug treatment while sensitivity to temozolomide was measured 12 days after drug treatment. Experiment Overall Design: For each of the 50 melanoma-derived cell lines, 1 flask of cells was grown to 80% confluence and harvested for RNA isolation. Cells were lysed in buffer with 1% β-mercaptoethanol and RNA isolated using Qiagen RNeasy-plus RNA isolation kit which included a step to eliminate genomic DNA. RNA concentration was measured and quality assessed using the NanoDrop ND100 spectrophotometer. RNA was reverse transcribed and biotin-labeled cRNA synthesized. The product was hybridized to the Affymetrix human genechip HU133 Plus2 according to manufacturer's instructions. Data was initially assessed for quality and a relative value for expression was calculated as the difference between the perfect match and the mismatch signal intensities using the Affymetrix GeneChip Operating System (GCOS). A detection call (present, absent or marginal) as well as a detection p-value was also calculated. Data was normalized by multiplying each signal by a scaling factor such that the mean signal intensity for each GeneChip was equal to 500. Scaling factors were all under 10.
Project description:Nine heterogeneous melanoma cell lines including D10 and WM115 were studied for cancer stem cell characteristics in vitro. D10 cell line was the only cell line expressing the cancer stem cell marker CD133. Thus, gene expression profiling on CD133+ and CD133- D10 cells was carried out using Affymetrix GeneChips Human Genome U133A 2.0.