Skip to main content
Download PDF
  • Published:

A fluorescence microscopy method for quantifying the detection of prostaglandin endoperoxide H synthase-1 and CD-41 in MEG-01 cells

Biological Procedures Online volume 3, pages 54–63 (2001)Cite this article

Abstract

In platelets, PGHS-1-dependant formation of thromboxane A2 is an important modulator of platelet function and a target for pharmacological inhibition of platelet function by aspirin. Since platelets are anucleated cells, we have used the immortalized human megakaryoblastic cell line MEG-01, which can be induced to differentiate into platelet-like structures upon addition of TPA as a model system to study PGHS-1 gene expression. Using a specific antibody to PGHS-1 we have developed a technique using immunofluorescence microscopy and analysis of multiple digital images to monitor PGHS-1 protein expression as MEG-01 cells were induced to differentiate by a single addition of TPA (1.6 × 10−8 M) over a period of 8 days. The method represents a rapid and economical alternative to flow cytometry. Using this technique we observed that TPA induced adherence of MEG-01 cells, and only the non-adherent TPA-stimulated cells demonstrated compromised viability. The differentiation of MEG-01 cells was evaluated by the expression of the platelet-specific cell surface antigen, CD-41. The latter was expressed in MEG-01 cells at the later stages of differentiation. We demonstrated a good correlation between PGHS-1 expression and the overall level of cellular differentiation of MEG-01 cells. Furthermore, PGHS-1 protein expression, which shows a consistent increase over the entire course of differentiation can be used as an additional and better index by which to monitor megakaryocyte differentiation.

References

  1. Smith WL, Laneuville O. 1994. Cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism. In Prostaglandin Inhibitors in Tumor Immunology and Immunotherapy. Harris JE, Braun D, Anderson K, editors CRC Press, London. 1–18.

    Google Scholar 

  2. Kujubu DA, Reddy ST, Fletcher BS, Herschman HR. Expression of the protein product of the prostaglandin synthase-2/TIS10 gene in mitogen-stimulated Swiss 3T3 cells. Journal of Biological Chemistry 1993;268:5425–5430.

    PubMed  CAS  Google Scholar 

  3. Kujubu DA, Fletcher BS, Varnum BC, Lim RW, Herschman HR. TIS10, a phorbol ester tumor promoter-inducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin synthase/cyclooxygenase homologue. Journal of Biological Chemistry 1991;266:12866–12872.

    PubMed  CAS  Google Scholar 

  4. DeWitt DL, El-Harith EA, Kraemer SA, Andrews MJ, Yao EF, Armstrong RL, Smith WL. The aspirin and heme-binding sites of ovine and murine prostaglandin endoperoxide synthases. Journal of Biological Chemistry 1990;265:5192–5198.

    PubMed  CAS  Google Scholar 

  5. Lecomte M, Laneuville O, Ji C, DeWitt DL, Smith WL. Acetylation of human prostaglandin endoperoxide synthase-2 (cyclooxygenase-2) by aspirin. Journal of Biological Chemistry 1994;269:13207–13215.

    PubMed  CAS  Google Scholar 

  6. Smith WL, Garavito RM, DeWitt DL. Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2. Journal of Biological Chemistry 1996;271:33157–33160.

    Article  PubMed  CAS  Google Scholar 

  7. Seibert K, Zhang Y, Leahy K, Hauser S, Masferrer J, Perkins W, Lee L, Isakson P. Pharmacological and biochemical demonstration of the role of cyclooxygenase 2 in inflammation and pain. Proc.Natl.Acad.Sci.USA 1994; 1:12013–12017.

    Article  Google Scholar 

  8. Mazur EM. Megakaryocytopoiesis and platelet production: a review. Experimental Hematology. 1987;15:340–350.

    PubMed  CAS  Google Scholar 

  9. Breton-Gorius J, Vainchenker W. Expression of platelet proteins during the in vitro and in vivo differentiation of megakaryocytes and morphological aspects of their maturation. Seminars in Hematology 1986;23:43–67.

    PubMed  CAS  Google Scholar 

  10. Tabilio A, Vainchenker W, Van Haecke D, Vinci G, Guichard J, Henri A, Reyes F, Breton-Gorius J. Immunological characterization of the leukemic megakaryocytic line at light and electron microscopic levels. Leukemia Research 1984;8:769–781.

    Article  PubMed  CAS  Google Scholar 

  11. Baatout S. Phorbol esters: useful tools to study megakaryocyte differentiation. Hematology and Cell Therapy 1998;40:33–39.

    PubMed  CAS  Google Scholar 

  12. Ogura M, Morishima Y, Ohno R, Kato Y, Hirabayashi N, Nagura H, Saito H. Establishment of a novel human megakaryoblastic leukemia cell line, MEG-01, with positive Philadelphia chromosome. Blood 1985;66:1384–1392.

    PubMed  CAS  Google Scholar 

  13. Ogura M, Morishima Y, Okumura M, Hotta T, Takamoto S, Ohno R, Hirabayashi N, Nagura H, Saito H. Functional and morphological differentiation induction of a human megakaryoblastic leukemia cell line (MEG-01s) by phorbol diesters. Blood 1988;72:49–60.

    PubMed  CAS  Google Scholar 

  14. McNicol A. 1996. Platelet preparation and estimation of functional responses. In Platelets: Apractical approach. S. Watson and Authi. KS, editors. Oxford University Press, Oxford. 1–26.

    Google Scholar 

  15. Mroske C, Plant MH, Franks DJ, Laneuville O. Characterization of prostaglandin endoperoxide H synthase-1 enzyme expression during differentiation of the megakaryocytic cell line MEG-01. Experimental Hematology. 2000;28:411–421. Available from: URL: http://www.elsevier.com/locate/exphem and http://www.sciencedirect.com.

    Article  PubMed  CAS  Google Scholar 

  16. Cavalloni G, Dane A, Piacibello W, Bruno S, Lamas E, Brechot C, Aglietta M. The involvement of human-nuc gene in polyploidization of K562 cell line. Experimental Hematology 2000;28:1432–1440.

    Article  PubMed  CAS  Google Scholar 

  17. Debili N, Issaad C, Masse JM, Guichard J, Katz A, Breton-Gorius J, Vainchenker W. Expression of CD34 and platelet glycoproteins during human megakaryocytic differentiation. Blood 1992;80:3022–3035.

    PubMed  CAS  Google Scholar 

  18. Debili N, Kieffer N, Nakazawa M, Guichard J, Titeux M, Cramer E, Breton-Gorius J, Vainchenker W. Expression of platelet glycoprotein Ib by cultured human megakaryocytes: ultrastructural localization and biosynthesis. Blood 1990;76:368–376.

    PubMed  CAS  Google Scholar 

  19. Takeuchi M, Kuno H, Saito H, Yoshida T, Ogura M, Takeuchi K. Platelet-like particles released by inhibition of DNA synthesis in the human megakaryoblastic cell line, MEG-01 and MEG-01s. Tissue Culture Research Communication 1995;14:165–175.

    Google Scholar 

  20. Takeuchi K, Satoh M, Kuno H, Yoshida T, Kondo H, Takeuchi M. Platelet-like particle formation in the human megakaryoblastic leukaemia cell lines, MEG-01 and MEG-01s. British Journal of Haematology 1998;100:436–444.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Road, K1H 8M5, Ottawa, Ontario, Canada

    Douglas J. Franks

  2. Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, K1H 8M5, Ottawa, Canada

    Cameron Mroske & Odette Laneuville

Authors
  1. Douglas J. Franks
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cameron Mroske
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Odette Laneuville
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Douglas J. Franks.

Additional information

Published: December 12, 2001

Rights and permissions

Reprints and permissions

About this article

Cite this article

Franks, D.J., Mroske, C. & Laneuville, O. A fluorescence microscopy method for quantifying the detection of prostaglandin endoperoxide H synthase-1 and CD-41 in MEG-01 cells. Biol Proced Online 3, 54–63 (2001). https://doi.org/10.1251/bpo23

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1251/bpo23

Indexing terms

Biological Procedures Online

ISSN: 1480-9222

Contact us