pp 1-14

Part of the Methods in Molecular Biology book series

Immunohistochemical Detection of Sphingosine-1-Phosphate and Sphingosine Kinase-1 in Human Tissue Samples and Cell Lines

  • Gary M. Reynolds
  • Barbara Visentin
  • Roger Sabbadini
Protocol

Abstract

Sphingosine-1-phosphate (S1P) and the enzyme primarily responsible for its production, sphingosine kinase-1 (SphK-1), are dysregulated in multiple human diseases including cancer, multiple sclerosis (MS), diabetes, neurological diseases, fibrosis, and certain pathologies associated with impaired angiogenesis such as age-related macular degeneration (AMD). Antibody-based techniques to identify and localize S1P and SphK-1 within cells and tissue specimens represent a powerful tool, not only to understand biological role of these molecules but also to validate these unique in-class targets in multiple state diseases. Consequently, the potential applications of these molecules for therapy and diagnostic purposes are currently under investigation. Here, we describe a new improved technique, Agitated Low Temperature Epitope Retrieval (ALTER) for staining procedures, to identify expression of S1P and SphK-1 in human frozen tissue samples. The challenges encountered in the process of localization in tissue samples of lipid molecules such as S1P are discussed.

Keywords:

Bioactive lipids S1P SPHK1 Anti-S1P antibody LT002 ALTER technique Immunohistochemistry Liver cancer 

References

  1. 1.
    Hannun YA, Bell RM (1993) The sphingomyelin cycle: a prototypic sphingolipid signaling pathway. Adv Lipid Res 25:27–41PubMedGoogle Scholar
  2. 2.
    Igarashi Y (1997) Functional roles of sphingosine, sphingosine-1-phosphate, and methylsphingosines: in regard to membrane sphingolipid signaling pathways. J Biochem 122:1080–1087CrossRefPubMedGoogle Scholar
  3. 3.
    Hait NC, Oskeritzian CA, Paugh SW, Milstien S, Spiegel S (2006) Sphingosine kinases, sphingosine-1-phosphate, apoptosis and diseases. Biochim Biophys Acta 1758:2016–2026CrossRefPubMedGoogle Scholar
  4. 4.
    Liu H, Sugiura M, Nava VE, Edsall LC, Kono K, Poulton S, Milstein S, Kohama S, Spiegel S (2000) Molecular cloning and functional characterization of a novel mammalian sphingosine kinase type 2 isoform. J Biol Chem 275:19513–19520CrossRefPubMedGoogle Scholar
  5. 5.
    Nava VE, Hobson JP, Murthy S, Milstien S, Speigel S (2002) Sphingosine kinase type 1 promotes estrogen-dependent tumorgenesis of breast cancer MCF-7 cells. Exp Cell Res 281:115–127CrossRefPubMedGoogle Scholar
  6. 6.
    Cuvillier O, Pirianov G, Kleuser B, Vanek PG, Coso OA, Gutkind S, Spiegel S (1996) Suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate. Nature 381:800–803ADSCrossRefPubMedGoogle Scholar
  7. 7.
    Chun J, Rosen H (2006) Lysophospholipid receptors as potential drug targets in tissue transplantation and autoimmune diseases. Curr Pharm Des 12:161–171CrossRefPubMedGoogle Scholar
  8. 8.
    Van Brocklyn JR, Lee M, Menzeleev R, Olivera A, Edsall L, Cuvillier O, Thomas DM, Coopman PJP, Thangada S, Liu CH, Hla T, Speigel S (1998) Dual actions of sphingosine- 1-phosphate: extracellular through the Gi-coupled receptor Edg-1 and intracellular to regulate proliferation and survival. J Cell Biol 142:229–240CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Pyne NJ, Pyne S (2010) Sphingosine-1-phosphate and cancer. Nat Rev Cancer 10:489–503CrossRefPubMedMATHGoogle Scholar
  10. 10.
    Ader I, Malavaud B, Cuvillier O (2009) When the sphingosine kinase 1/sphingosine-1-phosphate pathway meets hypoxia signaling: new targets for cancer therapy. Cancer Res 69(9):3723–3726CrossRefPubMedGoogle Scholar
  11. 11.
    Sabbadini RA (2010) Sphingosine-1-phosphate antibodies as potential agents in the treatment of cancer and age-related macular degeneration. Br J Pharmacol 162:1225–1238CrossRefGoogle Scholar
  12. 12.
    Xia P, Gamble J, Wang L, Pitson SM, Moretti PA, Wattenberg BW, D’Andrea RJ, Vadas MA (2000) An oncogenic role of sphingosine kinase. Curr Biol 10:1527–1530CrossRefPubMedGoogle Scholar
  13. 13.
    Vadas MA, Gamble JR (1996) Endothelial adhesion molecules in atherogenesis. Circ Res 79:1216–1217CrossRefPubMedGoogle Scholar
  14. 14.
    Johnson KR, Johnson KY, Crellin HG, Ogretmen B, Boylan AM, Harley RA, Obeid LM (2005) Immunohistochemical distribution of sphingosine kinase 1 in normal and tumor lung tissue. J Histochem Cytochem 59:1159–1166CrossRefGoogle Scholar
  15. 15.
    French KJ, Schrecengost RS, Lee BD, Zhuang Y, Smith SN, Eberly JL, Yun JK, Smith CD (2003) Discovery and evaluation of inhibitors of human sphingosine kinase. Cancer Res 63:5962–5969PubMedGoogle Scholar
  16. 16.
    Kawamori T, Osta W, Johnson KR, Pettus BJ, Bielawski J, Tanaka T, Wargovich MJ, Reddy BS, Hannun YA, Obeid LM, Zhou D (2006) Sphingosine kinase 1 is up-regulated in colon carcinogenesis. FASEB J 20:386–388PubMedGoogle Scholar
  17. 17.
    Shida D, Fang X, Kordula T, Takabe K, Lepine S, Alvarez SE, Milstien S, Spiegel S (2008) Cross-talk between LPA1 and epidermal growth factor receptors mediates up-regulation of sphingosine kinase 1 to promote gastric cancer cell motility and invasion. Cancer Res 68:6569–6577CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Long JS, Edwards J, Watson C, Tovey S, Mair KM, Schiff R, Natarajan V, Pyne NJ, Pyne S (2010) Sphingosine kinase 1 induces tolerance to human epidermal growth factor receptor 2 and prevents formation of a migratory phenotype in response to sphingosine-1-phosphate in estrogen receptor-positive breast cancer cells. Mol Cell Biol 30:3827–3841CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Li JY, Wang H, May S, Song X, Fueyo J, Fuller GN (2008) Constitutive activation of c-Jun N-terminal kinase correlates with histologic grade and EGFR expression in diffuse gliomas. J Neurooncol 88:11–17CrossRefPubMedGoogle Scholar
  20. 20.
    Ruckhaberle E, Rody A, Engels K, Gaetje R, von Minckwitz G, Schiffmann S, Grosch S, Geisslinger G, Holtrich U, Karn T, Kaufmann M (2008) Microarray analysis of altered sphingolipid metabolism reveals prognostic significance of sphingosine kinase 1 in breast cancer. Breast Cancer Res Treat 112:41–52CrossRefPubMedGoogle Scholar
  21. 21.
    Facchinetti MM, Gandini NA, Fermento ME, Sterin-Speziale NB, Ji Y, Patel V, Gutkind JS, Rivadulla MG, Curino AC (2010) The expression of sphingosine kinase-1 in head and neck carcinoma. Cells Tissues Organs 192(5):314–324CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Li W, Yu CP, Xia JT, Zhang L, Weng GX, Zheng HQ, Kong QL, Hu LJ, Zeng MS, Zeng YX, Li M, Li J, Song LB (2009) Sphingosine kinase 1 is associated with gastric cancer progression and poor survival of patients. Clin Cancer Res 15:1393–1399CrossRefPubMedGoogle Scholar
  23. 23.
    Van Brocklyn JR, Jackson CA, Pearl DK, Kotur MS, Snyder PJ, Prior TW (2005) Sphingosine kinase-1 expression correlates with poor survival of patients with glioblastoma multiforme: roles of sphingosine kinase isoforms in growth of glioblastoma cell lines. J Neuropathol Exp Neurol 64:695–705CrossRefPubMedGoogle Scholar
  24. 24.
    Malavaud B, Pchejetski D, Mazerolles C, de Paiva GR, Calvet C, Doumerc N, Pitson S, Rischmann P, Cuvillier O (2010) Sphingosine kinase-1 activity and expression in human prostate cancer resection specimens. Eur J Cancer 46:3417–3424CrossRefPubMedGoogle Scholar
  25. 25.
    Kapitonov D, Allegood JC, Mitchell C, Hait NC, Almenara JA, Adams JK, Zipkin RE, Dent P, Kordula T, Milstien S, Spiegel S (2009) Targeting sphingosine kinase 1 inhibits Akt signaling, induces apoptosis, and suppresses growth of human glioblastoma cells and xenografts. Cancer Res 69:6915–6923CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Paugh SW, Paugh BS, Rahmani M, Kapitonov D, Almenara JA, Kordula T, Milstien S, Adams JK, Zipkin RE, Grant S, Spiegel S (2008) A selective sphingosine kinase 1 inhibitor integrates multiple molecular therapeutic targets in human leukemia. Blood 112(4):1382–1391CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Pchejetski D, Doumerc N, Golzio M, Naymark M, Teissie J, Kohama T, Waxman J, Malavaud B, Cuvillier O (2008) Chemosensitizing effects of sphingosine kinase-1 inhibition in prostate cancer cell and animal models. Mol Cancer Ther 7:1836–1845CrossRefPubMedGoogle Scholar
  28. 28.
    Shen Z, Wu M, Elson P, Kennedy AW, Belinson J, Casey G, Xu Y (2001) Fatty acid composition of LPA and LPI in plasma from patients with ovarian cancer and other gynecological diseases. Gynecol Oncol 83:25–31CrossRefPubMedGoogle Scholar
  29. 29.
    Sutphen R, Xu Y, Wilbanks GD, Fiorica J, Grendys EC Jr, LaPolla JP, Arango H, Hoffman MS, Martino M, Wakeley K, Griffin D, Blanco RW, Cantor AB, Xiao YJ, Krischer JP (2004) Lysophospholipids are potential biomarkers of ovarian cancer. Cancer Epidemiol Biomarkers Prev 13:1185–1191Google Scholar
  30. 30.
    Xiao Y, Chen Y, Kennedy AW, Belinson J, Xu Y (2000) Evaluation of plasma lysophospholipids for diagnostic significance using electrospray ionization mass spectrometry (ESI-MS) analyses. Ann N Y Acad Sci 905:242–259ADSCrossRefPubMedGoogle Scholar
  31. 31.
    Xiao YJ, Schwartz B, Washington M, Kennedy A, Webster K, Belinson J, Xu Y (2001) Electrospray ionization mass spectrometry analysis of lysophospholipids in human ascitic fluids: comparison of the lysophospholipid contents in malignant vs nonmalignant ascitic fluids. Anal Biochem 290:302–313CrossRefPubMedGoogle Scholar
  32. 32.
    Xu Y, Shen Z, Wiper DW, Wu M, Morton RE, Elson P, Kennedy AW, Belinson J, Markman M, Casey G (1998) Lysophosphatidic acid as a potential biomarker for ovarian and other gynecologic cancers. JAMA 280:719–723CrossRefPubMedGoogle Scholar
  33. 33.
    Ammit AJ, Hastie AT, Edsall LC, Hoffman RK, Amrani Y, Krymskaya VP, Kane SA, Peters SP, Penn RB, Spiegel S, Panettieri RA Jr (2001) Sphingosine-1-phosphate modulates human airway smooth muscle cell functions that promote inflammation and airway remodeling in asthma. FASEB J 15:1212–1214PubMedGoogle Scholar
  34. 34.
    Kulakowska A, Zendzian-Piotrowska M, Baranowski M, Kononczuk T, Drozdowski W, Gorski J, Bucki R (2010) Intrathecal increase of sphingosine-1-phosphate at early stage multiple sclerosis. Neurosci Lett 477:149–152CrossRefPubMedGoogle Scholar
  35. 35.
    Deutschman D, Carstens J, Klepper R, Smith W, Page MT, Young TR, Gleason L, Nakajima N, Sabbadini R (2003) Predicting obstructive coronary artery disease using serum sphingosine-1-phosphate. Am Heart J 146:62–68CrossRefPubMedGoogle Scholar
  36. 36.
    Reynolds GM, Young FI, Young JA, Williams A, Rowlands DC (1994) Microwave oven antigen retrieval applied to the immunostaining of cytopathology specimens. Cytopathology 5:345–358CrossRefPubMedGoogle Scholar
  37. 37.
    Reynolds GM, Williams A, Gregory J, Rowlands DC (1995) Abolition of endogenous alkaline phosphatase activity by microwave oven heating. J Pathol 176(2):217CrossRefPubMedGoogle Scholar
  38. 38.
    Reynolds GM, Billingham LJ, Gray LJ, Flavell JR, Najafipour S, Crocker J, Nelson P, Young LS, Murray PG (2002) IL-6 expression by Hodgkin/Reed-Sternberg cells is associated with the presence of ‘B’ symptoms and failure to achieve complete remission in patients with advanced Hodgkin’s disease. Br J Haematol 118:1–7CrossRefGoogle Scholar
  39. 39.
    O’Brien N, Jones ST, Williams DG, Cunningham HB, Moreno K, Visentin B, Gentile A, Vekich J, Shestowsky W, Hiraiwa M, Matteo R, Cavalli A, Grotjahn D, Grant M, Hansen G, Campbell MA, Sabbadini R (2009) Production and characterization of monoclonal anti-sphingosine-1-phosphate antibodies. J Lipid Res 50:2245–2257CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Wojciak JM, Zhu N, Schuerenberg KT, Moreno K, Shestowsky WS, Hiraiwa M, Sabbadini R, Huxford T (2009) The crystal structure of sphingosine-1-phosphate in complex with a Fab fragment reveals metal bridging of an antibody and its antigen. Proc Natl Acad Sci U S A 106:17717–17722ADSCrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Gary M. Reynolds
    • 1
  • Barbara Visentin
    • 2
  • Roger Sabbadini
    • 3
  1. 1.Centre for Liver Research and NIHR Biomedical Research UnitUniversity of Birmingham and Queen Elizabeth Hospital BirminghamBirminghamUK
  2. 2.La Jolla Biologics IncSan DiegoUSA
  3. 3.Stanford University School of MedicineStanfordUSA

Personalised recommendations