Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells in vitro and in xenografts: Identification of apoptotic genes as targets for demethylation

Khaldoun Al-Romaih¹^,2 , Gino R Somers¹^,3 , Jane Bayani² , Simon Hughes⁶ , Mona Prasad² , Jean-Claude Cutz⁵ , Hui Xue⁴ , Maria Zielenska¹^,3 , Yuzhuo Wang⁴^,7 and Jeremy A Squire¹^,2^,4

¹Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada. M5G 1L5

²The Ontario Cancer Institute, Princess Margaret Hospital, Toronto, Canada. M5G 2M9

³Department of Pediatric Laboratory Medicine, Hospital for Sick Children, Toronto, Canada. M5G 1X8

⁴Department of Cancer Endocrinology, British Columbia Cancer Agency, Vancouver, Canada. V5Z 1L3

⁵Departments of Pathology & Molecular Medicine, and Laboratory Medicine, McMaster University, St. Joseph's Healthcare – Hamilton Regional Laboratory Medicine Program, Hamilton, Canada L8N 4A6

⁶Division of Tumor Biology, Institute of Cancer and Cancer Research, UK Clinical Centre, Barts and the London School of Medicine and Dentistry, John Vane Science Centre, Charterhouse Square, London, United Kingdom, EC1M 6BQ

⁷The Prostate Centre, Vancouver General Hospital, Vancouver, Canada, V6H 3Z6

author email corresponding author email

Cancer Cell International 2007, 7:14doi:10.1186/1475-2867-7-14


Published:	10 September 2007

Additional files

Additional file 1:

The effectiveness of demethylation following 72 hours treatment with 1 μM decitabine at the SNRPN gene locus. The imprinted SNRPN gene is located on human chromosome band 15q11.3 and alterations in DNA methylation at this locus are associated with individuals with the Prader-Willi and Angelman syndromes [64,65]. It was utilized in this study to confirm that decitabine treatment reduced DNA methylation in U2OS cells. A) Schematic figure of the SNRPN gene showing the probe location relative to NotI and XbaI cutting sites. NotI is a methyl-sensitive restriction endonuclease that will only cut its recognition sequence when unmethylated. B) Samples 1 and 2 are controls and samples 3 and 4 were treated with decitabine for 3-days. Left panel shows the autoradiogram of the restriction digest of DNA samples 1–4 on a 0.8% agarose gel. Right panel is Southern blot showing an increase of 63% of the 0.9 kb NotI product as a result of decitabine treatment (63 % loss of CpG methylation at the locus in U2OS).

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Additional file 2:

Effect of decitabine on U2OS xenografts size (raw data). Raw data measurements of 12 control tumors and decitabine treated tumors expressed as in tumor volumes estimated using this formula: volume (mm³) = (0.52) × (length mm) × (width mm) × (height mm). L = Length, W = width, H = height. SD = standard deviation.

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Additional file 3:

Expression fold change as detected in the Affy experiments for U2OS (U2OS1 and 2). All listed genes had change p-value < 0.0025 in the duplicate experiments. FC = fold change. I = increase.

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Additional file 4:

Methylation % detected by Pyro-Q-CpG (raw data). Multiple CpG positions were tested for each gene for all samples and indicated is the methylation percentage in all experiments. NT and (CT) = no-treatment (control). TR = decitabine treated. NHOst = Normal human osteoblasts.

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Additional file 5:

Additional methodology. Detailed description of the methods and materials used in the experimental approach.

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