Abstract
The effect of differentiation induction by a tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA) on a clonal human megakaryoblastic cell line, MEG-O1s, was investigated, and a prominent response was demonstrated. The cells became weakly adherent, developed conspicuous cytoplasmic blebs, and displayed mature megakaryocytic characteristics by light microscopy such as the development of azurophilic cytoplasmic granules and a mosaic pattern of oxyphilic patches, multiplication of nuclei, and enhancement of the PAS reaction and alpha-naphthyl acetate esterase staining. Ultrastructural studies demonstrated the development of prominent cytoplasmic blebs, budding of blebs, and multiplication of nuclei. Numerous granules with central nucleoids that are similar to alpha granules had developed as well as granules with high electron density and clearly demarcated zones. Surface marker analysis revealed a moderate increase in IgG Fc receptor levels and a profound decrease in C3 receptor sites. By an immunofluorescent technique using monoclonal and polyclonal antibodies, a dramatic change in the expression of several megakaryocyte-platelet- specific proteins was demonstrated. All the proteins that had been expressed before induction such as platelet glycoprotein (GP) IIb/IIIa, fibrinogen, von Willebrand factor, factor XIIIA, beta-thromboglobulin (beta-TG), and HLA class 1 antigen were profoundly enhanced after induction by TPA. Induction by TPA led to the expression of fibronectin and factor V, which were not detected on nontreated cells. An ultrastructural immunoperoxidase study demonstrated platelet GPIb and GPIIb/IIIa in both plasma membranes and protein synthesis areas such as perinuclear cisternae and endoplasmic reticulum after TPA induction. beta-TG was also observed in some cytoplasmic granules of TPA-treated cells. TPA remarkably increased the secretion of beta-TG into the culture medium of MEG-01s. Ploidy was also increased from 2C to 4C to 4C to 8C. Similar maturation of MEG-01s was induced by other phorbol diester analogues such as phorbol-12,13-dibutyrate, but not by phorbol itself. These results indicate that phorbol diester, TPA, can bring about differentiation and maturation of a human megakaryoblastic cell line (MEG-01s) and that MEG-01s cells will provide a useful model for studying megakaryocytic differentiation and numerous megakaryocyte- platelet-specific proteins.
