Inhibition of tubulin polymerization in megakaryocyte cell lines leads to polyploidization which affects the metabolism of actin

Megakaryocyte polyploidization responds to platelet demand and results from the lack of cytoplasmic separation while the nucleus keeps dividing. In normal telophase, the plane of the actin constriction ring is determined by the tubulin spindle. In order to investigate the role of tubulin in the megakaryocyte polyploidization, two cell lines with megakaryocyte properties (DAMI and HEL) were incubated for 3 days in the presence or absence of colchicine (10 ng/ml), an inhibitor of the tubulin spindle. As compared to control conditions, cells cultured in the presence of colchicine reveal an augmentation of cell size, the apparition of multilobed nuclei and an increase in the cytoplasm basophilia, suggesting a megakaryocyte morphology. Furthermore, when cells are cultured in the presence of colchicine, diameters measured by morphometry augment from 17.4 μm ± 1.7 to 34.5 μm ± 2.0 and from 27.3 μm ± 0.3 to 40.2 μm ± 0.6 for DAMI and HEL cell lines, respectively (p < 0.05 by t-test). After four days of culture in the presence of colchicine, cells undergo arrest proliferation. Ploidy measured by flow cytometry, shows that control cells predominantly diploid (2N) become polyploid with the appearance of 8N, 16N and 32N cells after addition of colchicine. Moreover, the endomitotic index {mean of (log₂ DNA content expressed in N)-l} increases significantly from 0.5 ± 0.1 to 1.2 ± 0.1 and from 0.6 ± 0.1 to 1.4 ± 0.0 after treatment with colchicine for the DAMI and HEL cell lines, respectively. To identify the nature of the molecules involved in this phenomenon, both forms of actin (monomeric, G- and polymerized F-) were evaluated by a DNase I inhibition assay. G-actin contents in pg per 10⁶ cells are 13.0 pg ± 2.8 (m ± SEM) and 1.0 pg ± 0.1 for unstimulated DAMI and HEL cells. F-actin contents per 10⁶ cells are 5.8 pg ± 1.5 and 0.1 pg ± 0.0 for DAMI and HEL cells. The addition of colchicine for four days of culture significantly increased the G-actin content (251% and 475% of controls) and F-actin content (170% and 619% of controls) for DAMI and HEL cell lines, respectively. In contrast, the G/F-actin ratio was not affected by colchicine. DAMI cells from each ploidy class were then sorted on an ELITE Coulter and assayed for actin content. While total actin, G-actin and F-actin per cell were augmented in polyploid cells cultured with colchicine, there was a reduction in G-, F- and total actin contents per diploid equivalent when cells become polyploid. In conclusion, these data suggest that inhibition of the tubulin spindle by colchicine induces polyploidization of megakaryocytes by a reduction of both forms of actin, possibly by preventing the actin constriction ring in the telophase.