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Cytotechnology

, Volume 63, Issue 1, pp 25–33 | Cite as

A multi-gene transcriptional profiling approach to the discovery of cell signature markers

  • Youichiro Wada
  • Dan Li
  • Anne Merley
  • Andrew Zukauskas
  • William C. Aird
  • Harold F. Dvorak
  • Shou-Ching ShihEmail author
Original Research

Abstract

A profile of transcript abundances from multiple genes constitutes a molecular signature if the expression pattern is unique to one cell type. Here we measure mRNA copy numbers per cell by normalizing per million copies of 18S rRNA and identify 6 genes (TIE1, KDR, CDH5, TIE2, EFNA1 and MYO5C) out of 79 genes tested as excellent molecular signature markers for endothelial cells (ECs) in vitro. The selected genes are uniformly expressed in ECs of 4 different origins but weakly or not expressed in 4 non-EC cell lines. A multi-gene transcriptional profile of these 6 genes clearly distinguishes ECs from non-ECs in vitro. We conclude that (i) a profile of mRNA copy numbers per cell from a well-chosen multi-gene panel can act as a sensitive and accurate cell type signature marker, and (ii) the method described here can be applied to in vivo cell fingerprinting and molecular diagnosis.

Keywords

Molecular signature Gene expression profiling mRNA copy numbers Endothelial cells 

Notes

Acknowledgments

Grants Supported in part by US Public Health Service grants HL-64402 and P01 CA92644.

References

  1. Aerts JL, Gonzales MI, Topalian SL (2004) Selection of appropriate control genes to assess expression of tumor antigens using real-time RT-PCR. Biotechniques 36:84–86Google Scholar
  2. Aird WC (2005) Spatial and temporal dynamics of the endothelium. J Thromb Haemost 3:1392–1406CrossRefGoogle Scholar
  3. Aird WC, Edelberg JM, Weiler-Guettler H, Simmons WW, Smith TW, Rosenberg RD (1997) Vascular bed-specific expression of an endothelial cell gene is programmed by the tissue microenvironment. J Cell Biol 138:1117–1124CrossRefGoogle Scholar
  4. Bas A, Forsberg G, Hammarstrom S, Hammarstrom ML (2004) Utility of the housekeeping genes 18S rRNA, beta-actin and glyceraldehyde-3-phosphate-dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes. Scand J Immunol 59:566–573CrossRefGoogle Scholar
  5. Bjarnadottir H, Jonsson JJ (2005) A rapid real-time qRT-PCR assay for ovine beta-actin mRNA. J Biotechnol 117:173–182CrossRefGoogle Scholar
  6. Boutet SC, Quertermous T, Fadel BM (2001) Identification of an octamer element required for in vivo expression of the TIE1 gene in endothelial cells. Biochem J 360:23–29CrossRefGoogle Scholar
  7. Bustin SA (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29:23–39CrossRefGoogle Scholar
  8. Chi JT, Chang HY, Haraldsen G, Jahnsen FL, Troyanskaya OG, Chang DS, Wang Z, Rockson SG et al (2003) Endothelial cell diversity revealed by global expression profiling. Proc Natl Acad Sci USA 100:10623–10628CrossRefGoogle Scholar
  9. Couffinhal T, Duplaa C, Moreau C, Lamaziere JM, Bonnet J (1994) Regulation of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in human vascular smooth muscle cells. Circ Res 74:225–234Google Scholar
  10. Davies MJ, Gordon JL, Gearing AJ, Pigott R, Woolf N, Katz D, Kyriakopoulos A (1993) The expression of the adhesion molecules ICAM-1, VCAM-1, PECAM, and E-selectin in human atherosclerosis. J Pathol 171:223–229CrossRefGoogle Scholar
  11. Deng DX, Spin JM, Tsalenko A, Vailaya A, Ben-Dor A, Yakhini Z, Tsao P, Bruhn L et al (2006) Molecular signatures determining coronary artery and saphenous vein smooth muscle cell phenotypes: distinct responses to stimuli. Arterioscler Thromb Vasc Biol 26:1058–1065CrossRefGoogle Scholar
  12. Dougher M, Terman BI (1999) Autophosphorylation of KDR in the kinase domain is required for maximal VEGF-stimulated kinase activity and receptor internalization. Oncogene 18:1619–1627CrossRefGoogle Scholar
  13. Eppihimer MJ, Wolitzky B, Anderson DC, Labow MA, Granger DN (1996) Heterogeneity of expression of E- and P-selectins in vivo. Circ Res 79:560–569Google Scholar
  14. Fadel BM, Boutet SC, Quertermous T (1998) Functional analysis of the endothelial cell-specific Tie2/Tek promoter identifies unique protein-binding elements. Biochem J 330(Pt 1):335–343Google Scholar
  15. Fonsatti E, Del Vecchio L, Altomonte M, Sigalotti L, Nicotra MR, Coral S, Natali PG, Maio M (2001) Endoglin: an accessory component of the TGF-beta-binding receptor-complex with diagnostic, prognostic, and bioimmunotherapeutic potential in human malignancies. J Cell Physiol 188:1–7CrossRefGoogle Scholar
  16. Gory S, Vernet M, Laurent M, Dejana E, Dalmon J, Huber P (1999) The vascular endothelial-cadherin promoter directs endothelial-specific expression in transgenic mice. Blood 93:184–192Google Scholar
  17. Haab BB, Dunham MJ, Brown PO (2001) Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions. Genome Biol 2:RESEARCH0004Google Scholar
  18. Haynes BF, Denning SM, Le PT, Singer KH (1990) Human intrathymic T cell differentiation. Semin Immunol 2:67–77Google Scholar
  19. Henninger DD, Panes J, Eppihimer M, Russell J, Gerritsen M, Anderson DC, Granger DN (1997) Cytokine-induced VCAM-1 and ICAM-1 expression in different organs of the mouse. J Immunol 158:1825–1832Google Scholar
  20. Ivanova NB, Dimos JT, Schaniel C, Hackney JA, Moore KA, Lemischka IR (2002) A stem cell molecular signature. Science 298:601–604CrossRefGoogle Scholar
  21. Kehrl JH, Riva A, Wilson GL, Thevenin C (1994) Molecular mechanisms regulating CD19, CD20 and CD22 gene expression. Immunol Today 15:432–436CrossRefGoogle Scholar
  22. Klein D (2002) Quantification using real-time PCR technology: applications and limitations. Trends Mol Med 8:257–260CrossRefGoogle Scholar
  23. Kuzu I, Bicknell R, Fletcher CD, Gatter KC (1993) Expression of adhesion molecules on the endothelium of normal tissue vessels and vascular tumors. Lab Invest 69:322–328Google Scholar
  24. Lemieux GA, Blumenkron F, Yeung N, Zhou P, Williams J, Grammer AC, Petrovich R, Lipsky PE et al (2007) The low affinity IgE receptor (CD23) is cleaved by the metalloproteinase ADAM10. J Biol Chem 282:14836–14844CrossRefGoogle Scholar
  25. Patterson C, Perrella MA, Hsieh CM, Yoshizumi M, Lee ME, Haber E (1995) Cloning and functional analysis of the promoter for KDR/flk-1, a receptor for vascular endothelial growth factor. J Biol Chem 270:23111–23118CrossRefGoogle Scholar
  26. Ramalho-Santos M, Yoon S, Matsuzaki Y, Mulligan RC, Melton DA (2002) “Stemness”: transcriptional profiling of embryonic and adult stem cells. Science 298:597–600CrossRefGoogle Scholar
  27. Sentenac A (1985) Eukaryotic RNA polymerases. CRC Crit Rev Biochem 18:31–90CrossRefGoogle Scholar
  28. Shih SC, Smith LE (2005) Quantitative multi-gene transcriptional profiling using real-time PCR with a master template. Exp Mol Pathol 79:14–22CrossRefGoogle Scholar
  29. Shih SC, Robinson GS, Perruzzi P, Calvo A, Desai K, Green J, Ali I, Smith LE et al (2002) Molecular profiling of angiogenesis markers. Am J Pathol 161:35–41Google Scholar
  30. Watt SM, Gschmeissner SE, Bates PA (1995) PECAM-1: its expression and function as a cell adhesion molecule on hemopoietic and endothelial cells. Leuk Lymphoma 17:229–244CrossRefGoogle Scholar
  31. Whitfield ML, George LK, Grant GD, Perou CM (2006) Common markers of proliferation. Nat Rev Cancer 6:99–106CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Youichiro Wada
    • 2
  • Dan Li
    • 1
  • Anne Merley
    • 1
  • Andrew Zukauskas
    • 1
  • William C. Aird
    • 2
  • Harold F. Dvorak
    • 1
  • Shou-Ching Shih
    • 1
    Email author
  1. 1.Center for Vascular Biological Research and Department of PathologyBeth Israel Deaconess Medical Center, Harvard Medical SchoolBostonUSA
  2. 2.Center for Vascular Biological Research and Division of Molecular and Vascular MedicineBeth Israel Deaconess Medical Center, Harvard Medical SchoolBostonUSA

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