Calcification

Saos-2 osteosarcoma model of Calcification/bone deposition

A human model of bone deposition/calcification using the Saos-2 osteosarcoma cell line treated with ascorbic acid and BGP was provided by Kim Summers and Margaret Davis.

The human osteosarcoma cell line SAOS2 was first established from an osteosarcoma isolated from an 11 year old female patient in 1973 [1]. The line has been commonly used to study extracellular matrix and mechanical mechanisms in both expression and mineralisation studies [2] although the utility of SAOS2 as a human model system for normal osteoblast formation and function has been debated. Varying cellular morphology and proliferation has been observed [3], but SAOS2 demonstrates physiological levels of multiple osteoblastic markers including, osteocalcin (OC), bone sialoprotein (BSP) and decorin (DCN) [2]. In addition, active levels of alkaline phosphatase (ALPL) and the capacity to mineralize render SAOS2 a useful model system in the study of osteoblast function and ECM formation [4].

This experiment was designed to determine promoter specificity and gene expression patterns of key regulators early and late in the mineralization process using this human model system. In addition, a non-calcifying human osteosarcoma cell line, MG63, was included in the study (time course data unavailable) as a control to identify mineralization independent effects [2][5].

The experiment was designed across 18 time points: 0, 15, 30, 45, 60, 80,100,120,150 and 180 minutes, 4, 8, and 24 hour as well as 4, 7, 14, 21, and 28 days following calcification induction. Cells were plated into 6 well plates (NUNC) at approximately 100,000 cells / well and mineralization was induced with 50µg/ml ascorbic acid (Sigma) and 2.5mM BGP (Sigma) following culture confluence. The 18 time point samples were isolated through the following methods. The cells were lifted using 1x Trypsin-EDTA solution (Sigma) and RNA was isolated using the RNA Bee protocol (Ambisco). The sample RNA was quantified using a Nanodrop spectrophotometer (Nanodrop, USA). Three biological replicates were performed.

Mineralization was verified using alizarin red staining of calcium at time point 0, 7, 14, 21 and 28 days. The cells were fixed with 4% paraformaldehyde (Sigma) for 10 minutes at room temperature, and rinsed with 1% PBS solution. The cells were stained using 1ml of 2% Alizarin Red (pH 4.2) for 5 minutes at room temperature. The wells were washed 3 times with dH20. The wells were extracted with 10% cetypyridium chloride and the extract used for quantification. The optical density was recorded using Multiskan ascent (Thermo) plate reader at a wavelength of 570nm.

The samples will be analyzed to develop a better understanding of the process and key regulating genes and promoter regions in ECM mineralization.

Zenbu configurations and status

• Saos-2 osteosarcoma treated with ascorbic acid and BGP(COMPLETED 18 timepoints in triplicate)

Gene expression profiles

• Known gene expression for the Saos-2 series (RefSeq based promoters)
  • TPM
  • tagcount

• Promoterome v1.0 expression for the Saos-2 series
• Top dynamic TFs in Saos-2 series
• OSC Cluster expression for the Saos-2 series
  • https://fantom5-collaboration.gsc.riken.jp/webdav/home/m.lizio/20111011_other_expression_tables_by_timecourse/human.Saos-2.hCAGE.hg19.tpm.L2clusters.osc.txt.gz

Motifs

• Top dynamic motifs in Saos-2 series

MARA based network results

• Saos-2 calcification network based on top 30 dynamic motifs
  • Most recent version (April 9, 2012):
    • https://fantom5-collaboration.gsc.riken.jp/webdav/home/arner/timecourse/release_120409/human_Saos-2_osteosarcoma/human_Saos-2_osteosarcoma.mara
  • Previous version(s):
    • https://fantom5-collaboration.gsc.riken.jp/webdav/home/m.lizio/20111011_other_expression_tables_by_timecourse/human.Saos-2.hCAGE.hg19.tpm.MARA.txt.gz

Self-organising maps

File:BGP.pdf

TSS Switching (Switch Engine)

• TSS dynamics plots can be found here:Media:saos2osteosarcoma_bgp_ascorbic acid.plots.pdf (it is recommended to download these files instead of viewing in browser as they are large in size)
• These figures only show those genes for which Switch Engine has detected TSS switching to occur. For now, only the first and last time points are being compared to define a switch.
  • One gene per page, RefSeq and Gene Symbol identifiers on top. Each panel is a separate TSS associated with the gene. TPM expression on y-axis and time on x-axis. Colored points correspond to specific expression, with colors referring to different replicates (key on top). Replicates with a * next to the name are those that have missing data from the currently available release of the DPI clustered and normalized TPM matrix. Such missing data were replaced by imputed values. Black lines are the mean trajectories across replicates. Purple vertical bars correspond to the time points being compared for switch definition. Magnitude of switch is expressed in each panel under TSS identifier with approximate 95% confidence interval in brackets. Note: some confidence intervals may not be accurate or may be missing due to the small sample size. TSS identifiers colored GREEN show an increasing TSS, those colored RED show decreasing TSS, WHITE show no statistically significant change.
  • TSS switch defined as the simultaneous presence of one or more increasing, together with one or more decreasing TSSs per gene. At least one increase and at least one decrease has to be statistically significant (alpha approximately 5%). At least one of the two time points being compared has to be greater than 5 tpm. At least 2 replicates have to agree to call an increasing/decreasing trajectory. TSSs have to be at least 250 base pairs away from one another to call a switch.
• A summary file can be found here: Media:saos2osteosarcoma_bgp_ascorbic acid.summary.xls
  • The file contains the following columns: RefSeq ID, Gene Symbol, TSS ID, X{value}: mean tpm expression at {value} time point, Delta: change in tpm expression from first to last time point, FC: fold change in tpm expression from first to last time point, DeltaSum: net change in tpm expression for this gene (summed across all TSS in this gene), Balance: ratio of increasing to decreasing tpm expression TSSs per gene (e.g. a balance value of 1 means that the total increase in tpm expression in increasing TSSs is equal to the total decrease in tpm expression in decreasing TSSs), Chromosome, Strand, Start position of TSS cluster, Stop position of TSS cluster, DBTSS: distance between TSSs relative to NA value.
• A list of genes in which TSS switching occurs can be found here: Media:saos2osteosarcoma_bgp_ascorbic acid.genelist.refseq.doc and Media:saos2osteosarcoma_bgp_ascorbic acid.genelist.symbol.doc
• BED file with regions corresponding to TSS clusters involved in TSS switching can be found here: Media:saos2osteosarcoma_bgp_ascorbic acid.switchinglist.bed.xls (please remove .xls extension after downloading)

Related samples

• Osteosarcoma cell lines 143B/TK^(-)neo^(R) and HS-Os-1 from RIKEN BRC - contact Al Forrest
• Additional untreated timecourse is stored
• Additional timecourse using MG63 is stored

Quality control

Most recent version (April 9 2012):

https://fantom5-collaboration.gsc.riken.jp/webdav/home/arner/timecourse/release_120409/human_Saos-2_osteosarcoma/

Previous version(s):

File:Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification.zip

• Staining pictures...

References

• First paper on Saos-2: http://www.ncbi.nlm.nih.gov/pubmed/3040234

1. Fogh, J., J.M. Fogh, and T. Orfeo, One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice. J Natl Cancer Inst, 1977. 59(1): p. 221-6.

2. Pautke, C., et al., Characterization of osteosarcoma cell lines MG-63, Saos-2 and U-2 OS in comparison to human osteoblasts. Anticancer Res, 2004. 24(6): p. 3743-8.

3. Boskey, A.L. and R. Roy, Cell culture systems for studies of bone and tooth mineralization. Chem Rev, 2008. 108(11): p. 4716-33.

4. McQuillan, D.J., et al., Matrix Deposition by a Calcifying Human Osteogenic Sarcoma Cell Line (SAOS-2).Bone, April 1995. 16(4):p.415-26

5. Billiau, A., et al., Human interferon: mass production in a newly established cell line, MG-63. Antimicrob Agents Chemother, 1977. 12(1): p. 11-5.