Aphidicolin, an inhibitor of DNA replication, blocks the TPA-induced differentiation of a human megakaryoblastic cell line, MEG-O1

Abstract

The commitment process of a human megakaryoblastic cell line (MEG-O1) induced with phorbol ester, TPA, was investigated with special reference to glycoprotein (GP) IIb/IIIa expression, multinuclear formation, and DNA replication. TPA (10(-7) mol/L) completely inhibited cellular division in MEG-O1, but did not suppress de novo DNA synthesis. Two days' culture with 10(-7) mol/L TPA was sufficient for MEG-O1 cells to initiate an irreversible commitment process. These cells could not resume cell growth and expressed GP IIb/IIIa antigen; some of them showed multinuclear form and DNA polyploidy even after removal of TPA from the culture medium. DNA histogram analysis showed that, upon treatment with TPA, the percentage of cells whose DNA ploidy was more than 8N was 5 to 10 times higher than that of control cells. Precise analysis using cell size fractionation by centrifugal elutriation method showed that there was strong correlation between the percentage of multinuclear cells and DNA polyploidy in TPA-treated cells. The percentage and staining intensity of GP IIb/IIIa and other megakaryocytic phenotypes such as von Willebrand factor and PAS staining were highest in large multinuclear cell populations, suggesting that these cells are the most differentiated population in this system. In TPA-treated cells, the activity of DNA polymerase alpha, a marker for cell growth, remained at the same level as in control cells. Aphidicolin, a specific inhibitor of DNA polymerase alpha, completely inhibited the differentiation induction of MEG-O1 cells with TPA measured by either GP IIb/IIIa expression or multinuclear cell formation. Therefore, DNA replication appears to be involved in the process of phenotypic expression as well as endomitosis in megakaryocyte differentiation of MEG-O1 cells. Aphidicolin was also effective in inhibiting megakaryocytic differentiation of other leukemia cell lines such as human erythroleukemia (HEL) and K562 cell lines induced with TPA, suggesting the close interplay of DNA replication and phenotypic expression in megakaryopoiesis.

Activin A and Retinoic Acid Synergize in Cyclooxygenase‐1 and Thromboxane Synthase Induction During Differentiation of J774.1 Macrophages

“…The long incubation time of 48 h required for maximal effect indicates that cell division may be a prerequisite for activin-A action. Indeed, aphidicolin, an inhibitor of DNA polymerase a [31] and thereby of DNA replication, also blocked the activin-A-dependent induction, suggesting cell-division-dependent differentiation of the J774.1 cells as a cause for the increase in cyclooxygenase activity, in agreement with the slow time response ( Fig. 3).…”

Platelet‐like particle formation in the human megakaryoblastic leukaemia cell lines, MEG‐01 and MEG‐01s

“…The clonal human megakaryoblastic leukaemia cell line MEG‐01 and its subline MEG‐01s, established by Ogura et al (1985 , 1988), display phenotypic properties that closely resemble those of megakaryoblasts but not other blood cell lineages. These properties demonstrate increased expression after treatment of the cells with phorboldiester, TPA ( Murate et al , 1991 ). The cell lines also release particles identified by a characteristic marginal microtubule and by the localization of platelet‐specific glycoprotein (GPIIb/IIIa) in the plasma membrane ( Takeuchi et al , 1991 , 1995).…”

K‐252a‐induced polyploidization and differentiation of a human megakaryocytic cell line, Meg‐J: transient elevation and subsequent suppression of cyclin B1 and cdc2 expression in the process of polyploidization

“…K‐252a induced Meg‐J cells to form polyploid cells, which were accompanied by the morphological differentiation and increases in the levels of GPIIb/IIIa and GPIb expression. It has been suggested that DNA synthesis is a prerequisite not only for polyploidization but also for the expression of megakaryocytic markers such as GPs in TPA‐treated megakaryocytic differentiation ( Murata et al , 1991 , 1993; Yoshino et al , 1996 ). In our system, such DNA synthesis without cell division may also have induced increased levels of megakaryocytic markers.…”

“…Phorbol‐12‐myristate‐13‐acetate (TPA) was reported to induce megakaryocytic differentiation in some megakaryocytic cell lines. However, its ability to induce polyploidization was not sufficient to allow analysis of the mechanism of polyploidization ( Ogura et al , 1988 ; Long et al , 1990 ; Greenberg et al , 1988 ; Murata et al , 1991 ). K‐252a, an indolocarbasole derivative protein kinase inhibitor isolated from the culture broth of Nocardiosis SP ( Kase et al , 1986 , 1987) causes polyploidization without intervening mitosis in various cultured cell lines ( Usui et al , 1991 ).…”