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)
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.
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.
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.
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.
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.
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.
Smith WL, Garavito RM, DeWitt DL. Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2. Journal of Biological Chemistry 1996;271:33157–33160.
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.
Mazur EM. Megakaryocytopoiesis and platelet production: a review. Experimental Hematology. 1987;15:340–350.
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.
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.
Baatout S. Phorbol esters: useful tools to study megakaryocyte differentiation. Hematology and Cell Therapy 1998;40:33–39.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Published: December 12, 2001
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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