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Series GSE7454 Query DataSets for GSE7454
Status Public on Apr 05, 2007
Title Modulation of gene expression by decitabine in U-2OS cells in vitro and in vivo
Organism Homo sapiens
Experiment type Expression profiling by array
Methylation profiling by array
Summary Background: Epigenetic modifications such as methylation silencing of genes with CpG-island-associated promoters is frequently observed in cancer. Studies regarding the implications of epigenetic modifications in osteosarcoma (OS) have been limited. The epigenetic drug decitabine is a potential re-activator of silenced genes through de-methylation, and is currently undergoing clinical trials for cancer treatment. No study to date has utilized decitabine to modify gene expression in OS-derived cells to identify gene-specific methylation targets that may have therapeutic importance. The objective of this study was to measure the response of the OS cell line, U-2OS, to decitabine treatment both in vitro and in vivo.
Results: Genome-wide screening was used to distinguish decitabine-dependent changes in gene expression in U-2OS and to identify responsive loci with de-methylated CpG regions. U-2OS xenografts were established in the sub-renal capsule of immune-deficient mice to study the effect of decitabine in vivo on tumor growth and differentiation. Genome-wide screening of U-2OS cells in vitro revealed that decitabine treatment at low-dosage (1 µM) caused a significant induction (p<0.0025) in the expression of 88 genes, 13 had a ≥2-fold change, 11 of which had CpG-island-associated promoters. Interestingly, 6 of these 11 were pro-apoptotic genes. 1 µM decitabine resulted in a significant induction of cell death in U-2OS cells in vitro (p<0.05). Decitabine treatment also reduced tumor volume size significantly (p<0.05) in U-2OS in vivo at a 2.5 mg/kg dose. Histological analysis of U-2OS xenograft sections revealed a lower mitotic activity (p<0.0001), a higher bone matrix production (p<0.0001), and higher apoptosis (p = 0.0329) after treatment. The 6 pro-apoptotic genes were also induced to ≥2-fold in vivo. Quantitative Methylation Pyrosequencing (Pyro Q-CpG) showed that 4 pro-apoptotic genes had CpG-island DNA de-methylation as a result of treatment in vitro and in vivo.
Conclusion: Our data provide new insights regarding the use of epigenetic modifiers in OS, and have important implications for therapeutic trials involving demethylation drugs. The findings of this study suggest an epigenetic mechanism, whereby OS tumor cells silence expression of pro-apoptotic genes through promoter-CpG methylation, to maintain high proliferation capacity and continuous growth.
Keywords: Osteosarcoma, epigenetics, methylation, decitabine
Overall design Total RNA was extracted using the RNeasy kit (Qiagen, Germany) from duplicate experiments of U-2OS cells at Day3 after treatment with 1 µM decitabine or medium alone (control). In each experiment RNA yields were pooled from two independent cultures per treatment arm to minimize experimental noise. For each case, 10 µg of RNA was labeled and hybridized to the Affymetrix HG-U133A GeneChips using the manufacturer’s protocol (Affymetrix, Santa Clara, CA) by the Center of Applied Genomics at the Hospital for Sick Children (Toronto, Canada). Data were extracted using the Microarray Suite (MAS) version 5.0 (Affymetrix) and linearly scaled to achieve an average intensity of 150 across each chip. The candidate gene list obtained from the MAS 5.0-extracted data was selected by eliminating genes that were not present in at least one experiment. The arrays were subjected to a pair wise comparison using MAS 5.0, with signal intensities from the no-treatment cells as the baseline. The statistical significance for the change of expression for each probe set between the decitabine treated and control was calculated by the MAS5.0 software. The criteria for gene selection for real-time expression validation analysis was based on the statistically significant up-regulation (p<0.0025) and fold change of ≥ 2 for expression after decitabine treatment. The gene list was annotated based on the NetAffx data-base ( /index.affx) and further verified using the Human Genome Browser data base (
Contributor(s) Al Romaih KI, Prasad M, Zielenska M, Squire JA
Citation(s) 17845729
Submission date Apr 04, 2007
Last update date Mar 25, 2019
Contact name Khaldoun Al-Romaih
Phone 416 946 4509
Organization name Ontario Cancer Institute
Department AMO
Lab Dr Squire Lab
Street address 610 University Ave
City Toronot
ZIP/Postal code M5G 2M9
Country Canada
Platforms (2)
GPL96 [HG-U133A] Affymetrix Human Genome U133A Array
GPL570 [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array
Samples (4)
GSM180457 U-2OS (no treatment) expression profile (AffyChip U133A platform)
GSM180459 U-2OS (no-treatment) expression profile (AffyChip U133 Plus 2.0 platform)
GSM180461 U-2OS (decitabine treated) expression profile (AffyChip U133A platform)
BioProject PRJNA100213

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Supplementary file Size Download File type/resource
GSE7454_RAW.tar 17.5 Mb (http)(custom) TAR (of CEL, EXP)

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