Funes et al. 10.1073/pnas.0700690104. |
Fig. 6. Heatmaps of GEM data showing the expression of genes known to be downstream of oncogenes introduced into MSC. When E7 is expressed, genes downstream of pRB inactivation are expressed, including E2F-responsive and DNA replication genes. Genes which are down-regulated when p53 is inactivated are shown when E6 is expressed. Genes affected when ST is expressed are shown, as well as genes associated with the Ras pathway. These data support the validity of the array experiments. 0-5 indicates parental MSC (0), and each subsequent step in transformation. Red indicates up-regulation, and blue down-regulation from the mean.
Fig. 7. Heatmaps show the effect of MSC transformation on selected genes involved in apoptotic, angiogenic, immune and metastatic pathways obtained by performing array experiments in triplicate using Affymetrix U133 Plus 2.0 chips. Full gene lists are available in SI Table 4. Lists for each 'hallmark' were compiled from the Affymetrix (www.affymetrix.com) and SuperArray (www.superarray.com) databases, and expression data analyzed for each list separately. As expected, anti-apoptotic pathways are up-, and pro-apoptotic pathways down-regulated during transformation, especially after the introduction of E6 (p53 inactivation). The up-regulation of angiogenic genes, including angiopoietin 2 (ANGPT2), ephrin B2 (EFNB2) and fibroblast growth factor 1 (FGF1) (red arrows), is associated with the introduction of ST (c-Myc stabilization). ST and c-Myc are known to down-regulate MHC class I molecules (1, 2) and we show here a striking down-regulation of both class I and II molecules (blue and gray arrows respectively) after the introduction of ST. GEM also shows genes involved in invasion and metastasis of tumor cells. Of note, ezrin (villin 2, VIL2, black arrow), associated with the metastatic ability and poor prognosis of osteosarcoma (3), is highly up-regulated after the introduction of ST, linking ezrin transcription to PP2A inactivation. This could provide another mechanism for promotion of tumor metastasis by c-Myc.
Fig. 8. (A) Overall expression of MSC markers SH2, SH4 and Stro-1 was maintained during the step-wise introduction of oncogenic 'hits'. Markers considered to be negative for MSC, such as CD45 and CD34, were not expressed in 5 'hits' MSC. Control cells are also shown (black). (B) Lipid accumulation during adipogenic differentiation (+) was assessed with Oil Red O staining and cells were visualized by light microscopy (1x or 2x). MSC transduced with 4 and 5 'hits' maintained their ability to undergo differentiation toward fat (adipocytes). Small fat droplets (arrows) can be seen after the introduction of exogenous genes (4 and 5 'hits'). Control cells, where adipogenesis was not induced (-), are also shown. Adipogenic markers are also expressed during MSC transformation. Transcription factors involved in adipogenesis such as aP2, C/EBPa and PPARg were detected by RT-PCR although there was less expression of C/EBPa and PPARg in the 4 and 5 'hits' than in parental (0) MSC. (C) Chondrogenic differentiation in 5 'hits' MSC was assessed with toluidine blue staining. Pellet size (left) and a section of stained pellet (right) are shown in cells in which chondrogenesis was (+) or was not (-) induced. The pellet-volume ratio in 5 'hits' MSC (±) is shown. Pellet volume was calculated using the standard formula: a x b2 ´ 0.52 where a is the longest diameter and b is the shortest diameter. Pellet sections were photographed at x100 magnification.
Fig. 9. Schematic representation of genes involved in glycolysis, the PPP, the TCA cycle and nucleotide biosynthesis (a representative probe for each gene was chosen). This figure was based on GenMAPP software (4), Gladstone Institutes, UCSF. Red indicates up-regulation and blue down-regulation from the mean expression. For each gene a heatmap strip summarizes the average of three different samples. Labels 0 to 5 above each heatmap strip show the different steps ('hits') used for transformation. In addition, we show four unsummarized heatmaps with all of the probes for each gene. The pathways for glycolysis and the TCA cycle show no overall significant change during progressive transformation (P = 0.2715 and P = 0.1157 respectively). However, the heatmaps for the PPP and nucleotide metabolism show a significant overall change (P = 0.0039 and P < 0.0001 respectively) during step-wise transformation. All probe-sets were included for the statistical analysis (see Supporting Materials and Methods).
Fig. 10. (A) Human fibroblast (HF) cell lines were named according to the number of steps or 'hits' introduced sequentially by retroviral transduction (0-5). Cells were probed by antibodies against introduced oncogenes or their downstream cellular targets after retroviral infection as previously described for MSC in Fig. 2B. (B) Anchorage-independent growth at different stages of HF transformation (1 and 5 'hits') is shown. (C) Tumor formation (arrows) in nude mice inoculated with 5 'hits' HF is also shown.
1. Versteeg R, Noordermeer IA, Krusewolters M, Ruiter DJ, Schrier PI (1988) EMBO J 7:1023-1029.
2. Moreno CS, Ramachandran S, Ashby DG, Laycock N, Plattner CA, Chen W, Hahn WC, Pallas DC (2004) Cancer Res 64:6978-6988.
3. Khanna C, Wan XL, Bose S, Cassaday R, Olomu O, Mendoza A, Yeung C, Gorlick R, Hewitt SM, Helman LJ (2004) Nat Med 10:182-186.
4. Dahlquist KD, Salomonis N, Vranizan K, Lawlor SC, Conklin BR (2002) Nat Genet 31:19-20.
SI Table 3
Probe Set ID Gene Title UniGene ID Gene Symbol
202275_at glucose-6-phosphate dehydrogenase Hs.461047 G6PD
1555685_at Hexose-6-phosphate dehydrogenase (glucose 1-dehydrogenase) Hs.463511 H6PD
206933_s_at hexose-6-phosphate dehydrogenase (glucose 1-dehydrogenase) Hs.463511 H6PD
221892_at hexose-6-phosphate dehydrogenase (glucose 1-dehydrogenase) Hs.463511 H6PD
226160_at hexose-6-phosphate dehydrogenase (glucose 1-dehydrogenase) Hs.463511 H6PD
201118_at phosphogluconate dehydrogenase /// phosphogluconate dehydrogenase Hs.464071 PGD
1554316_at 6-phosphogluconolactonase Hs.466165 PGLS
218387_s_at 6-phosphogluconolactonase Hs.466165 PGLS
218388_at 6-phosphogluconolactonase Hs.466165 PGLS
230699_at 6-phosphogluconolactonase Hs.466165 PGLS
212973_at ribose 5-phosphate isomerase A (ribose 5-phosphate epimerase) Hs.469264 RPIA
201463_s_at transaldolase 1 Hs.438678 TALDO1
208699_x_at transketolase (Wernicke-Korsakoff syndrome) Hs.89643 TKT
208700_s_at transketolase (Wernicke-Korsakoff syndrome) Hs.89643 TKT
228205_at transketolase (Wernicke-Korsakoff syndrome) Hs.89643 TKT
SI Table 4
Probe Set ID Gene Title UniGene ID Gene Symbol
1552721_a_at fibroblast growth factor 1 (acidic) Hs.483635 FGF1
1552939_at angiopoietin 1 Hs.369675 ANGPT1
1553573_s_at ephrin-A2 Hs.532655 EFNA2
1554828_at "platelet-derived growth factor receptor, alpha polypeptide" Hs.74615 PDGFRA
1554997_a_at prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) Hs.196384 PTGS2
1555319_at stabilin 1 Hs.301989 STAB1
1555320_a_at stabilin 1 Hs.301989 STAB1
1555468_at neuropilin 2 Hs.471200 NRP2
1555540_at "transforming growth factor, beta 3" Hs.2025 TGFB3
1555952_at "Collagen, type XVIII, alpha 1" Hs.517356 COL18A1
1555953_at "collagen, type XVIII, alpha 1" Hs.517356 COL18A1
1556836_at TIMP metallopeptidase inhibitor 2 Hs.104839 TIMP2
1556837_a_at TIMP metallopeptidase inhibitor 2 Hs.104839 TIMP2
1557910_at "heat shock 90kDa protein 1, beta" Hs.509736 HSPCB
1559360_at "Nuclear RNA-binding protein, putative" Hs.128518 EFNA5
1559921_at platelet/endothelial cell adhesion molecule (CD31 antigen) Hs.514412 PECAM1
1559975_at "B-cell translocation gene 1, anti-proliferative" Hs.255935 BTG1
1560657_at Tyrosine kinase with immunoglobulin-like and EGF-like domains 1 Hs.78824 TIE1
1561365_at Neuropilin 1 Hs.131704 NRP1
1561889_at "Pleiotrophin (heparin binding growth factor 8, neurite growth-promoting factor 1)" Hs.371249 PTN
1566677_at "Matrix metallopeptidase 2 (gelatinase A, 72kDa gelatinase, 72kDa type IV collagenase)" Hs.513617 MMP2
1566678_at "Matrix metallopeptidase 2 (gelatinase A, 72kDa gelatinase, 72kDa type IV collagenase)" Hs.513617 MMP2
1568732_at "Collagen, type XVIII, alpha 1" Hs.517356 COL18A1
1569203_at chemokine (C-X-C motif) ligand 2 Hs.75765 CXCL2
1569583_at Epiregulin Hs.115263 EREG
200017_at ribosomal protein S27a /// ribosomal protein S27a Hs.311640 RPS27A
200064_at "heat shock 90kDa protein 1, beta /// heat shock 90kDa protein 1, beta" Hs.509736 HSPCB
200801_x_at "actin, beta" Hs.520640 ACTB
200878_at endothelial PAS domain protein 1 Hs.468410 EPAS1
200879_s_at endothelial PAS domain protein 1 Hs.468410 EPAS1
200920_s_at "B-cell translocation gene 1, anti-proliferative" Hs.255935 BTG1
200921_s_at "B-cell translocation gene 1, anti-proliferative" Hs.255935 BTG1
200989_at "hypoxia-inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor)" Hs.509554 HIF1A
201069_at "matrix metallopeptidase 2 (gelatinase A, 72kDa gelatinase, 72kDa type IV collagenase)" Hs.513617 MMP2
201107_s_at thrombospondin 1 Hs.164226 THBS1
201108_s_at thrombospondin 1 Hs.164226 THBS1
201109_s_at thrombospondin 1 Hs.164226 THBS1
201110_s_at thrombospondin 1 Hs.164226 THBS1
201147_s_at "TIMP metallopeptidase inhibitor 3 (Sorsby fundus dystrophy, pseudoinflammatory)" Hs.297324 TIMP3
201148_s_at "TIMP metallopeptidase inhibitor 3 (Sorsby fundus dystrophy, pseudoinflammatory)" Hs.297324 TIMP3
201149_s_at "TIMP metallopeptidase inhibitor 3 (Sorsby fundus dystrophy, pseudoinflammatory)" Hs.297324 TIMP3
201150_s_at "TIMP metallopeptidase inhibitor 3 (Sorsby fundus dystrophy, pseudoinflammatory)" Hs.297324 TIMP3
201666_at TIMP metallopeptidase inhibitor 1 Hs.522632 TIMP1
201808_s_at endoglin (Osler-Rendu-Weber syndrome 1) Hs.76753 ENG
201809_s_at endoglin (Osler-Rendu-Weber syndrome 1) Hs.76753 ENG
201891_s_at beta-2-microglobulin Hs.534255 B2M
202023_at ephrin-A1 Hs.516664 EFNA1
202273_at "platelet-derived growth factor receptor, beta polypeptide" Hs.509067 PDGFRB
202283_at "serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 1" Hs.532768 SERPINF1
202351_at "integrin, alpha V (vitronectin receptor, alpha polypeptide, antigen CD51)" Hs.436873 ITGAV
202509_s_at "tumor necrosis factor, alpha-induced protein 2" Hs.525607 TNFAIP2
202510_s_at "tumor necrosis factor, alpha-induced protein 2" Hs.525607 TNFAIP2
202668_at ephrin-B2 Hs.149239 EFNB2
202669_s_at ephrin-B2 Hs.149239 EFNB2
202859_x_at interleukin 8 Hs.624 IL8
202888_s_at "alanyl (membrane) aminopeptidase (aminopeptidase N, aminopeptidase M, microsomal aminopeptidase, CD13, p150)" Hs.1239 ANPEP
202894_at EPH receptor B4 Hs.437008 EPHB4
203083_at thrombospondin 2 Hs.371147 THBS2
203084_at "transforming growth factor, beta 1 (Camurati-Engelmann disease)" Hs.1103 TGFB1
203085_s_at "transforming growth factor, beta 1 (Camurati-Engelmann disease)" Hs.1103 TGFB1
203131_at "platelet-derived growth factor receptor, alpha polypeptide" Hs.74615 PDGFRA
203167_at TIMP metallopeptidase inhibitor 2 Hs.104839 TIMP2
203683_s_at vascular endothelial growth factor B Hs.78781 VEGFB
203915_at chemokine (C-X-C motif) ligand 9 Hs.77367 CXCL9
203934_at kinase insert domain receptor (a type III receptor tyrosine kinase) Hs.479756 KDR
203936_s_at "matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV collagenase)" Hs.297413 MMP9
204053_x_at phosphatase and tensin homolog (mutated in multiple advanced cancers 1) Hs.500466 PTEN
204054_at phosphatase and tensin homolog (mutated in multiple advanced cancers 1) Hs.500466 PTEN
204150_at stabilin 1 Hs.301989 STAB1
204200_s_at platelet-derived growth factor beta polypeptide (simian sarcoma viral (v-sis) oncogene homolog) Hs.1976 PDGFB
204379_s_at "fibroblast growth factor receptor 3 (achondroplasia, thanatophoric dwarfism)" Hs.1420 FGFR3
204380_s_at "fibroblast growth factor receptor 3 (achondroplasia, thanatophoric dwarfism)" Hs.1420 FGFR3
204406_at fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor) Hs.507621 FLT1
204421_s_at fibroblast growth factor 2 (basic) Hs.284244 FGF2
204422_s_at fibroblast growth factor 2 (basic) Hs.284244 FGF2
204468_s_at tyrosine kinase with immunoglobulin-like and EGF-like domains 1 Hs.78824 TIE1
204470_at "chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha)" Hs.789 CXCL1
204533_at chemokine (C-X-C motif) ligand 10 Hs.413924 CXCL10
204574_s_at matrix metallopeptidase 19 Hs.154057 MMP19
204575_s_at matrix metallopeptidase 19 Hs.154057 MMP19
204582_s_at "kallikrein 3, (prostate specific antigen)" Hs.171995 KLK3
204583_x_at "kallikrein 3, (prostate specific antigen)" Hs.171995 KLK3
204625_s_at "integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61)" Hs.218040 ITGB3
204626_s_at "integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61)" Hs.218040 ITGB3
204627_s_at "integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61)" Hs.218040 ITGB3
204628_s_at "integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61)" Hs.218040 ITGB3
204642_at "endothelial differentiation, sphingolipid G-protein-coupled receptor, 1" Hs.154210 EDG1
204648_at natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide receptor A) Hs.490330 NPR1
204677_at "cadherin 5, type 2, VE-cadherin (vascular epithelium)" Hs.76206 CDH5
204748_at prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) Hs.196384 PTGS2
204858_s_at endothelial cell growth factor 1 (platelet-derived) Hs.546251 ECGF1
205015_s_at "transforming growth factor, alpha" Hs.170009 TGFA
205016_at "transforming growth factor, alpha" Hs.170009 TGFA
205067_at "interleukin 1, beta" Hs.126256 IL1B
205117_at fibroblast growth factor 1 (acidic) Hs.483635 FGF1
205127_at prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase) Hs.201978 PTGS1
205128_x_at prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase) Hs.201978 PTGS1
205141_at "angiogenin, ribonuclease, RNase A family, 5 /// ribonuclease, RNase A family, 4" Hs.283749 ANG /// RNASE4
205207_at "interleukin 6 (interferon, beta 2)" Hs.512234 IL6
205226_at platelet-derived growth factor receptor-like Hs.458573 PDGFRL
205247_at Notch homolog 4 (Drosophila) Hs.436100 NOTCH4
205463_s_at platelet-derived growth factor alpha polypeptide Hs.376032 PDGFA
205479_s_at "plasminogen activator, urokinase" Hs.77274 PLAU
205572_at angiopoietin 2 Hs.553484 ANGPT2
205608_s_at angiopoietin 1 Hs.369675 ANGPT1
205609_at angiopoietin 1 Hs.369675 ANGPT1
205611_at "tumor necrosis factor (ligand) superfamily, member 12" --- TNFSF12
205754_at coagulation factor II (thrombin) Hs.76530 F2
205767_at epiregulin Hs.115263 EREG
206083_at brain-specific angiogenesis inhibitor 1 Hs.194654 BAI1
206254_at epidermal growth factor (beta-urogastrone) Hs.419815 EGF
206295_at interleukin 18 (interferon-gamma-inducing factor) Hs.83077 IL18
206309_at leukocyte cell derived chemotaxin 1 Hs.421391 LECT1
206336_at chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2) Hs.164021 CXCL6
206390_x_at platelet factor 4 (chemokine (C-X-C motif) ligand 4) Hs.81564 PF4
206702_at "TEK tyrosine kinase, endothelial (venous malformations, multiple cutaneous and mucosal)" Hs.89640 TEK
206742_at c-fos induced growth factor (vascular endothelial growth factor D) Hs.11392 FIGF
206801_at natriuretic peptide precursor B Hs.219140 NPPB
206943_at "transforming growth factor, beta receptor I (activin A receptor type II-like kinase, 53kDa)" Hs.494622 TGFBR1
207092_at "leptin (obesity homolog, mouse)" Hs.194236 LEP
207113_s_at "tumor necrosis factor (TNF superfamily, member 2)" Hs.241570 TNF
207160_at "interleukin 12A (natural killer cell stimulatory factor 1, cytotoxic lymphocyte maturation factor 1, p35)" Hs.673 IL12A
207163_s_at v-akt murine thymoma viral oncogene homolog 1 Hs.525622 AKT1
207301_at ephrin-A5 Hs.128518 EFNA5
207351_s_at SH2 domain protein 2A Hs.103527 SH2D2A
207433_at interleukin 10 Hs.193717 IL10
207442_at colony stimulating factor 3 (granulocyte) Hs.2233 CSF3
207826_s_at "inhibitor of DNA binding 3, dominant negative helix-loop-helix protein" Hs.76884 ID3
207850_at chemokine (C-X-C motif) ligand 3 Hs.89690 CXCL3
207852_at chemokine (C-X-C motif) ligand 5 Hs.89714 CXCL5
208042_at angiogenic factor with G patch and FHA domains 1 Hs.213393 AGGF1
208173_at "interferon, beta 1, fibroblast" Hs.93177 IFNB1
208240_s_at fibroblast growth factor 1 (acidic) Hs.483635 FGF1
208256_at ephrin-A2 Hs.532655 EFNA2
208375_at "interferon, alpha 1" Hs.37026 IFNA1
208408_at "pleiotrophin (heparin binding growth factor 8, neurite growth-promoting factor 1)" Hs.371249 PTN
208417_at fibroblast growth factor 6 Hs.166015 FGF6
208937_s_at "inhibitor of DNA binding 1, dominant negative helix-loop-helix protein" Hs.504609 ID1
208981_at platelet/endothelial cell adhesion molecule (CD31 antigen) Hs.514412 PECAM1
208982_at Platelet/endothelial cell adhesion molecule (CD31 antigen) Hs.514412 PECAM1
208983_s_at platelet/endothelial cell adhesion molecule (CD31 antigen) Hs.514412 PECAM1
209035_at midkine (neurite growth-promoting factor 2) Hs.82045 MDK
209081_s_at "collagen, type XVIII, alpha 1" Hs.517356 COL18A1
209082_s_at "collagen, type XVIII, alpha 1" Hs.517356 COL18A1
209097_s_at jagged 1 (Alagille syndrome) Hs.224012 JAG1
209098_s_at jagged 1 (Alagille syndrome) Hs.224012 JAG1
209099_x_at jagged 1 (Alagille syndrome) Hs.224012 JAG1
209465_x_at "pleiotrophin (heparin binding growth factor 8, neurite growth-promoting factor 1)" Hs.371249 PTN
209466_x_at "pleiotrophin (heparin binding growth factor 8, neurite growth-promoting factor 1)" Hs.371249 PTN
209540_at insulin-like growth factor 1 (somatomedin C) Hs.160562 IGF1
209541_at insulin-like growth factor 1 (somatomedin C) Hs.160562 IGF1
209542_x_at insulin-like growth factor 1 (somatomedin C) Hs.160562 IGF1
209652_s_at "placental growth factor, vascular endothelial growth factor-related protein" Hs.252820 PGF
209747_at "transforming growth factor, beta 3" Hs.2025 TGFB3
209774_x_at chemokine (C-X-C motif) ligand 2 Hs.75765 CXCL2
209909_s_at "transforming growth factor, beta 2" Hs.133379 TGFB2
209946_at vascular endothelial growth factor C Hs.435215 VEGFC
209960_at hepatocyte growth factor (hepapoietin A; scatter factor) Hs.396530 HGF
209961_s_at hepatocyte growth factor (hepapoietin A; scatter factor) Hs.396530 HGF
209977_at plasminogen Hs.558366 PLG
209978_s_at "lipoprotein, Lp(a) /// plasminogen" Hs.520120 LPA /// PLG
210132_at ephrin-A3 Hs.516656 EFNA3
210133_at chemokine (C-C motif) ligand 11 Hs.54460 CCL11
210150_s_at "laminin, alpha 5" Hs.244352 LAMA5
210163_at chemokine (C-X-C motif) ligand 11 Hs.518814 CXCL11
210287_s_at fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor) Hs.507621 FLT1
210354_at "interferon, gamma" Hs.856 IFNG
210510_s_at neuropilin 1 Hs.131704 NRP1
210512_s_at vascular endothelial growth factor Hs.73793 VEGF
210513_s_at vascular endothelial growth factor Hs.73793 VEGF
210615_at neuropilin 1 Hs.131704 NRP1
210710_at angiogenic factor with G patch and FHA domains 1 Hs.213393 AGGF1
210755_at hepatocyte growth factor (hepapoietin A; scatter factor) Hs.396530 HGF
210841_s_at neuropilin 2 Hs.471200 NRP2
210842_at neuropilin 2 Hs.471200 NRP2
210997_at hepatocyte growth factor (hepapoietin A; scatter factor) Hs.396530 HGF
210998_s_at hepatocyte growth factor (hepapoietin A; scatter factor) Hs.396530 HGF
211122_s_at chemokine (C-X-C motif) ligand 11 Hs.518814 CXCL11
211148_s_at angiopoietin 2 Hs.553484 ANGPT2
211258_s_at "transforming growth factor, alpha" Hs.170009 TGFA
211506_s_at interleukin 8 Hs.624 IL8
211527_x_at vascular endothelial growth factor Hs.73793 VEGF
211533_at "platelet-derived growth factor receptor, alpha polypeptide" Hs.74615 PDGFRA
211577_s_at insulin-like growth factor 1 (somatomedin C) Hs.160562 IGF1
211579_at "integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61)" Hs.218040 ITGB3
211668_s_at "plasminogen activator, urokinase /// plasminogen activator, urokinase" Hs.77274 PLAU
211711_s_at phosphatase and tensin homolog (mutated in multiple advanced cancers 1) /// phosphatase and tensin homolog (mutated in multiple advanced cancers 1) Hs.500466 PTEN
211737_x_at "pleiotrophin (heparin binding growth factor 8, neurite growth-promoting factor 1) /// pleiotrophin (heparin binding growth factor 8, neurite growth-promoting factor 1)" Hs.371249 PTN
211844_s_at neuropilin 2 Hs.471200 NRP2
212171_x_at vascular endothelial growth factor Hs.73793 VEGF
212298_at neuropilin 1 Hs.131704 NRP1
213201_s_at "troponin T type 1 (skeletal, slow)" Hs.534085 TNNT1
213867_x_at "actin, beta" Hs.520640 ACTB
214081_at plexin domain containing 1 Hs.125036 PLXDC1
214359_s_at "heat shock 90kDa protein 1, beta" Hs.509736 HSPCB
214632_at neuropilin 2 Hs.471200 NRP2
214641_at "collagen, type IV, alpha 3 (Goodpasture antigen)" Hs.471525 COL4A3
214974_x_at chemokine (C-X-C motif) ligand 5 Hs.89714 CXCL5
215101_s_at chemokine (C-X-C motif) ligand 5 Hs.89714 CXCL5
215179_x_at "Placental growth factor, vascular endothelial growth factor-related protein" Hs.252820 PGF
215305_at "platelet-derived growth factor receptor, alpha polypeptide" Hs.74615 PDGFRA
215775_at Thrombospondin 1 Hs.164226 THBS1
215813_s_at prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase) Hs.201978 PTGS1
216055_at Platelet-derived growth factor beta polypeptide (simian sarcoma viral (v-sis) oncogene homolog) Hs.1976 PDGFB
216061_x_at platelet-derived growth factor beta polypeptide (simian sarcoma viral (v-sis) oncogene homolog) Hs.1976 PDGFB
216231_s_at beta-2-microglobulin Hs.534255 B2M
216261_at "integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61)" Hs.218040 ITGB3
216268_s_at jagged 1 (Alagille syndrome) Hs.224012 JAG1
216367_at "collagen, type IV, alpha 3 (Goodpasture antigen)" Hs.471525 COL4A3
216368_s_at "collagen, type IV, alpha 3 (Goodpasture antigen)" Hs.471525 COL4A3
216598_s_at chemokine (C-C motif) ligand 2 Hs.303649 CCL2
216680_s_at EPH receptor B4 Hs.437008 EPHB4
216867_s_at platelet-derived growth factor alpha polypeptide Hs.376032 PDGFA
216893_s_at "collagen, type IV, alpha 3 (Goodpasture antigen)" Hs.471525 COL4A3
216896_at "collagen, type IV, alpha 3 (Goodpasture antigen)" Hs.471525 COL4A3
216898_s_at "collagen, type IV, alpha 3 (Goodpasture antigen)" Hs.471525 COL4A3
217112_at platelet-derived growth factor beta polypeptide (simian sarcoma viral (v-sis) oncogene homolog) Hs.1976 PDGFB
217497_at endothelial cell growth factor 1 (platelet-derived) Hs.546251 ECGF1
218368_s_at "tumor necrosis factor receptor superfamily, member 12A" Hs.355899 TNFRSF12A
218534_s_at angiogenic factor with G patch and FHA domains 1 Hs.213393 AGGF1
219257_s_at sphingosine kinase 1 Hs.68061 SPHK1
219367_s_at Neuropilin 2 Hs.471200 NRP2
219403_s_at heparanase Hs.44227 HPSE
219700_at plexin domain containing 1 Hs.125036 PLXDC1
219803_at angiopoietin-like 3 Hs.209153 ANGPTL3
220114_s_at stabilin 2 Hs.408249 STAB2
220183_s_at nudix (nucleoside diphosphate linked moiety X)-type motif 6 Hs.558459 NUDT6
220406_at "transforming growth factor, beta 2" Hs.133379 TGFB2
220407_s_at "transforming growth factor, beta 2" Hs.133379 TGFB2
220480_at heart and neural crest derivatives expressed 2 Hs.388245 HAND2
221009_s_at angiopoietin-like 4 Hs.9613 ANGPTL4
221085_at "tumor necrosis factor (ligand) superfamily, member 15" Hs.241382 TNFSF15
222006_at "Fibroblast growth factor receptor 3 (achondroplasia, thanatophoric dwarfism)" Hs.1420 FGFR3
222033_s_at Fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor) Hs.507621 FLT1
222073_at "collagen, type IV, alpha 3 (Goodpasture antigen)" Hs.471525 COL4A3
222176_at phosphatase and tensin homolog (mutated in multiple advanced cancers 1) Hs.500466 PTEN
222661_at angiogenic factor with G patch and FHA domains 1 Hs.213393 AGGF1
222877_at Neuropilin 2 Hs.471200 NRP2
222881_at heparanase Hs.44227 HPSE
223333_s_at angiopoietin-like 4 Hs.9613 ANGPTL4
223510_at neuropilin 2 Hs.471200 NRP2
224560_at TIMP metallopeptidase inhibitor 2 Hs.104839 TIMP2
224594_x_at "actin, beta" Hs.520640 ACTB
224793_s_at "transforming growth factor, beta receptor I (activin A receptor type II-like kinase, 53kDa)" Hs.494622 TGFBR1
225363_at Phosphatase and tensin homolog (mutated in multiple advanced cancers 1) Hs.500466 PTEN
225566_at neuropilin 2 Hs.471200 NRP2
226497_s_at Fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor) Hs.507621 FLT1
226498_at Fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor) Hs.507621 FLT1
227469_at Phosphatase and tensin homolog (mutated in multiple advanced cancers 1) Hs.500466 PTEN
228006_at Phosphatase and tensin homolog (mutated in multiple advanced cancers 1) Hs.500466 PTEN
228102_at Neuropilin 2 Hs.471200 NRP2
228103_s_at Neuropilin 2 Hs.471200 NRP2
228121_at "Transforming growth factor, beta 2" Hs.133379 TGFB2
228586_at Endoglin (Osler-Rendu-Weber syndrome 1) Hs.76753 ENG
228699_at Neuropilin 2 Hs.471200 NRP2
228778_at "Microcephaly, primary autosomal recessive 1" Hs.490892 MCPH1
229225_at neuropilin 2 Hs.471200 NRP2
229570_at "CDNA FLJ25011 fis, clone CBL01244" Hs.473256 ---
229830_at Platelet-derived growth factor alpha polypeptide Hs.376032 PDGFA
229904_at Endothelial PAS domain protein 1 Hs.468410 EPAS1
229924_s_at Jagged 1 (Alagille syndrome) Hs.224012 JAG1
230101_at Chemokine (C-X-C motif) ligand 2 Hs.75765 CXCL2
230329_s_at nudix (nucleoside diphosphate linked moiety X)-type motif 6 Hs.558459 NUDT6
230410_at Neuropilin 2 Hs.471200 NRP2
230562_at "Microcephaly, primary autosomal recessive 1" Hs.490892 MCPH1
230711_at Endothelial PAS domain protein 1 Hs.468410 EPAS1
231183_s_at Jagged 1 (Alagille syndrome) Hs.224012 JAG1
231579_s_at TIMP metallopeptidase inhibitor 2 Hs.104839 TIMP2
231629_x_at "Kallikrein 3, (prostate specific antigen)" Hs.171995 KLK3
231684_at Angiopoietin-like 3 Hs.209153 ANGPTL3
232311_at Beta-2-microglobulin Hs.534255 B2M
232406_at Jagged 1 (Alagille syndrome) Hs.224012 JAG1
232629_at prokineticin 2 Hs.528665 PROK2
232701_at Neuropilin 2 Hs.471200 NRP2
232809_s_at Fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor) Hs.507621 FLT1
233254_x_at phosphatase and tensin homolog (mutated in multiple advanced cancers 1) Hs.500466 PTEN
233314_at phosphatase and tensin homolog (mutated in multiple advanced cancers 1) Hs.500466 PTEN
233626_at Neuropilin 1 Hs.131704 NRP1
233701_at Neuropilin 1 Hs.131704 NRP1
234458_at "Alanyl (membrane) aminopeptidase (aminopeptidase N, aminopeptidase M, microsomal aminopeptidase, CD13, p150)" Hs.1239 ANPEP
234576_at "Alanyl (membrane) aminopeptidase (aminopeptidase N, aminopeptidase M, microsomal aminopeptidase, CD13, p150)" Hs.1239 ANPEP
235086_at Thrombospondin 1 Hs.164226 THBS1
235963_at Endothelial PAS domain protein 1 Hs.468410 EPAS1
236034_at Angiopoietin 2 Hs.553484 ANGPT2
236561_at "Transforming growth factor, beta receptor I (activin A receptor type II-like kinase, 53kDa)" Hs.494622 TGFBR1
236678_at Jagged 1 (Alagille syndrome) Hs.224012 JAG1
237567_at stabilin 2 Hs.408249 STAB2
237843_at Endothelial PAS domain protein 1 Hs.468410 EPAS1
237958_at "Microcephaly, primary autosomal recessive 1" Hs.490892 MCPH1
238327_at similar to MGC52679 protein --- LOC440836
238669_at prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase) Hs.201978 PTGS1
238869_at "Hypoxia-inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor)" Hs.509554 HIF1A
239336_at Thrombospondin 1 Hs.164226 THBS1
239519_at Neuropilin 1 Hs.131704 NRP1
239605_x_at "Transforming growth factor, beta receptor I (activin A receptor type II-like kinase, 53kDa)" Hs.494622 TGFBR1
240033_at plasminogen Hs.558366 PLG
240171_at Prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase) Hs.201978 PTGS1
240243_at Fibroblast growth factor 2 (basic) Hs.284244 FGF2
240738_at Neuropilin 1 Hs.131704 NRP1
240786_at Notch homolog 4 (Drosophila) Hs.436100 NOTCH4
240963_x_at Plexin domain containing 1 Hs.125036 PLXDC1
240964_at Phosphatase and tensin homolog (mutated in multiple advanced cancers 1) Hs.500466 PTEN
241119_at Angiopoietin 1 Hs.369675 ANGPT1
242203_at Midkine (neurite growth-promoting factor 2) Hs.82045 MDK
242214_at ribosomal protein S27a /// similar to bA92K2.2 (similar to ubiquitin) /// similar to ribosomal protein S27a Hs.311640 RPS27A /// LOC388720 /// LOC389425
242622_x_at Phosphatase and tensin homolog (mutated in multiple advanced cancers 1) Hs.500466 PTEN
242677_at Neuropilin 1 Hs.131704 NRP1
242868_at Endothelial PAS domain protein 1 Hs.468410 EPAS1
243799_x_at Angiopoietin-like 3 Hs.209153 ANGPTL3
244399_at Stabilin 2 Hs.408249 STAB2
244624_at ribosomal protein S27a /// similar to bA92K2.2 (similar to ubiquitin) /// similar to ribosomal protein S27a Hs.311640 RPS27A /// LOC388720 /// LOC389425
32625_at natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide receptor A) Hs.490330 NPR1
38487_at stabilin 1 Hs.301989 STAB1
39402_at "interleukin 1, beta" Hs.126256 IL1B
SI Materials and Methods
Adipogenic differentiation.
Briefly, cells were seeded at 25 ´ 103 cells per cm2 in Mesencult medium containing 10% serum supplemented with 1 ng ml-1 bFGF. One day postconfluence, adipogenic differentiation was induced via addition of medium without bFGF, supplemented with 1 mM dexamethasone, 500 mM IBMX, 200 mM indomethacin (all from Sigma) and 10 mg ml-1 insulin (Roche). The medium was changed every 2 days. Cells were fixed in 4% formaldehyde for 30 min, washed and stained for 2 h with 0.06% Oil Red O dissolved in 60% triethyl phosphate. Excess dye was removed and cells were visualized by light microscopy.For RT-PCR analysis, total RNA was extracted from differentiating MSC using RNeasy Mini Kit (Qiagen, Sussex, U.K.). cDNA was synthesized from 0.1 mg total RNA using Superscript II reverse transcriptase (Invitrogen, Paisley, U.K.), and used as a template for PCR under the following conditions: 94°C for 2 min, (n) cycles of 94°C for 30 seconds, 55°C for 1 min, 72°C for 45 seconds, then a 72°C incubation for 10 min. Cycle number and primer sequences were:
PPARg: 5'-TTCAGAAATGCCTTGCAGTG-3' 5'-CACCTCTTTGCTCTGCTCCT-3' (28)x
C/EBPa: 5'-GAGGAGGGGAGAATTCTTGG-3' 5'-CCCTATGTTTCCACCCCTTT-3' (28)x
aP2: 5'-TGCAGCTTCCTTCTCACCTT-3' 5'-CATCCCCATTCACACTGATG-3' 25x
b-actin: 5'-CCAAGGCCAACCGCGAGAAGATGAC-3' 5'AGGGTACATGGTGGTGC CGCCAGAC-3' (25)x
Osteogenic differentiation.
Cells were seeded at 2 ´ 104 per cm2 and cultured in Mesencult medium containing 10% serum without bFGF, and supplemented with 10 mM b-glycerophosphate, 100 nM dexamethasone, and 50 mg ml-1 ascorbic acid. Medium was changed every 3 days and cells assessed for osteogenic differentiation after 14 days. Cells were fixed with 10% formalin/PBS for 10 min and stained with AS-MX phosphate alkaline phosphate solution with fast blue RR salt for 45 min. Finally, cells were rinsed in distilled H2O to remove excess dye.Chondrogenic differentiation.
Cells (2.5 ´ 105) were suspended in 500 m l of chondrocyte differentiation medium (high glucose DMEM with 100 nM dexamethasone, 50 mg ml-1 ascorbate-2-phosphate, 40 mg ml-1 L-proline, 100 mg ml-1 sodium pyruvate, 50 mg ml-1 ITS+premix (Invitrogen: 6.25 mg ml-1 insulin, 6.25 mg ml-1 transferrin, 6.25 ng ml-1 selenious acid, 1.25 mg ml-1 BSA, 5.35 mg ml-1 linoleic acid), 500 ng ml-1 BMP6 (R&D systems) and 10 ng ml-1 TGFb3 (R&D systems)). The suspension was placed in a 15 ml falcon tube and centrifuged at 600 ´ g for 30 min. The cells formed a loose pellet at the bottom of the tube. After 4 days, and thereafter every 3 days, the medium was replaced. Pellet cultures were assessed for extracellular matrix production after 21 days. For toluidine blue staining, cell pellets from cartilage differentiation were fixed overnight at 4°C in 4% paraformaldehyde dissolved in 2X PBS. Fixed pellets were dehydrated by sequential ethanol and xylene treatment and finally embedded in paraffin wax. Wax sections were cut (5 mm), dewaxed and rehydrated with sequential xylene and ethanol incubations. Pellet sections were incubated for 45 seconds in toluidine blue stain (0.1% wt/vol toluidine blue dissolved in 0.1 M sodium phosphate buffer), and excess stain removed by distilled H2O washing. Sections were dehydrated by consecutive ethanol and xylene washes before mounting with DPX, and photographed at x100 magnification.Tissue samples and array processing.
Gene expression microarrays were performed on each MSC cell line in triplicate (Supporting Fig. 6). All experiments with these cells used Affymetrix Hg U133 Plus 2.0 chip arrays. Five mouse tumors were also used for gene expression microarray analyses, but here we used Affymetrix Hg U133av2 chips to compare the expression profiles against those previously generated from 96 human mesenchymal tumors (1). RNA was extracted and cDNA probes labeled as previously described (1, 2) following the manufacturer's recommendation using an Affymetrix GeneChip Instrument System (see URL below).Microarray Analysis.
Data analysis was carried out using the R statistical environment and programming language (3). We extensively used R software packages from Bioconductor (4), an open source bioinformatics resource. We used the 'affy' package written to handle Affymetrix data, and specifically the 'rma' algorithm for preprocessing, normalizing and calculation of expression values (5, 6). Heatmaps were created using the Cluster and Treeview software packages (7). For a comparison of MSC cell lines (Hg U133 Plus 2.0) with mouse tumors (Hg U133av2) we quantile normalized the expression values of the intersecting probesets after 'rma' preprocessing.Multidimensional Scaling and Geneset Enrichment Analysis.
For comparison of mouse tumors with a sarcoma database (Hg U133av2) we used the classical MDS method (or principle coordinates method) and a subset of genes previously identified as a 'molecular fingerprint' for sarcoma (1). For gene enrichment analysis of metabolic pathways we calculated all probeset t-statistics between MSC and MSC 5 'hits'. We then resampled a set of t-statistics equal to the size of the test pathway 10000 times from the data. The p-value was calculated as the proportion of times the average resampling t-statistic was greater than the average pathway t-statistic. Note that all t-statistics were made positive to perform a two-sided significance test (8).URLs.
Information on Affymetrix U133 oligonucleotide arrays is available at http://www.affymetrix.com/products/arrays/specific/hgu133.affx. Information on the biotinylated target RNA preparation is available at http://www.affymetrix.com/support/technical/manual/expression_manual.affx. The ArrayExpress database is available at http://www.ebi.ac.uk/arrayexpress. The Bioconductor suite of software is available at http://www.bioconductor.org. Information on the R statistical programming language is available at http://www.r-project.org.Accession numbers.
Details of quality control measures and all microarray data can be found in the ArrayExpress database with the accession numbers: E-MEXP-562 for MSC tumors and E-MEXP-563 for MSC transformation.1. Henderson SR, Guiliano D, Presneau N, McLean S, Frow R, Vujovic S, Anderson J, Sebire N, Whelan J, Athanasou N, Flanagan AM, Boshoff C (2005) Genome Biol 6:R76.1-R76.11.
2. Wang HW, Trotter MW, Lagos D, Bourboulia D, Henderson S, Makinen T, Elliman S, Flanagan AM, Alitalo K, Boshoff C (2004) Nat Genet 36:687-693.
3. Venables WN, Ripley BD. (2002) Modern Applied Statistics with S (Springer, New York).
4. Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge YC, Gentry J, et al. (2004) Genome Biol 5:
5. Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP. (2003) Biostatistics 4:249-264.
6. Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP (2003) Nucleic Acids Res 31:e15.
7. Eisen MB, Spellman PT, Brown PO, Botstein D (1998) Proc Natl Acad Sci USA 95:14863-14868.
8. Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, Lehar J, Puigserver P, Carlsson E, Ridderstrale M, Laurila E, et al. (2003) Nat Genet 34:267-273.