Initiation of DNA synthesis by microtubule disruption in quiescent rat 3Y1 cells

Abstract

Disruption of cytoplasmic microtubules by colchicine, colcemid or vinblastine induced the initiation of DNA synthesis and cell division in quiescent cultures of rat embryo fibroblast cell line, 3Y1, in the absence of growth factors. The microtubule-disruption-induced DNA synthesis was so marked that we could initiate analysis of the mechanism. Incubation of quiescent 3Y 1 cells with high concentrations of vinblastine or those of vinblastine plus colcemid, which formed large tubulin paracrystals concomitant with the depolymerization of cytoplasmic microtubules, initiated DNA synthesis in the cells. Because these treatments did not increase the free tubulin level in the cells, but rather decreased it, an increase in the free tubulin level may not be required for initiation of DNA synthesis induced by microtubule-disrupting agents. Culture fluid of the 3Y1 cells incubated with colchicine did not stimulate the initiation of DNA synthesis when added to other quiescent 3Y1 cells after conversion of the colchicine in thc culture fluid to lumicolchicine by ultraviolet irradiation. This suggested that colchicine treatment did not induce secretion of any mitogenic factors from 3Y1 cells. These results suggest that disruption of normal microtubules may elicit intracellular signals leading to cell proliferation.Cytoplasmic microtubules function in a wide variety of ways as a structural component and motility machinery. Cytoplasmic microtubules have also been suggested to play a regulatory role in cell growth, based on the observation that microtubule-depolymerizing agents have inhibitory or stimulatory effects on cell cycle traverse of quiescent cultures. Vasiliev et al. reported that colcemid stimulated the initiation of DNA synthesis in primary cultures of density-arrested mouse embryo fibroblast-like cells in the presence of 10% bovine serum [I]. On the other hand, colchicine inhibited the initiation of DNA synthesis in some cell lines [2][3][4][5][6][7][8]. But, as for confluently cultured, well-arrested fibroblastic cells, colchicine always potentiated the effect of growth factors on initiation of DNA synthesis [9-131. Crossin and Carney demonstrated that colchicine treatment alone initiated DNA synthesis in somc primary cultures in serum-free meclium [14, IS], though the stimulation of thymidine incorporation by colchicine was no more than 2-_?-fold, probably because the cultures might not be completely rendered quiescent.We have found in this study that microtubule-disrupting agents in the absence of any growth factors induce effectively the initiation of DNA synthesis in quiescent cultures of the rat embryo fibroblast cell line, 3Y1. Using this system we could further investigate how microtubule depolymerization relates to the induction of DNA synthesis. Recent excellent experiments done by Cleveland and co-workers [16, 171 and others [I 81 have established that cytoplasmic free tubulin concentration could be a signal for regulating the level of tubulinCorrespondence to E. Nishida, Department of Biophysics and ...

Rapid reorganization of microtubular cytoskeleton accompanies early changes in nuclear ploidy and chromatin structure in postmitotic cells of barley leaves infected with powdery mildew

“…Similarly, colchicine was found to promote the growth-factor-driven cell cycle traverse in animal cells (Teng et al 1977). Transient disintegration of the MT cytoskeleton was reported by several authors to be necessary for the completion of the G1/S transition and subsequent initiation of DNA synthesis in non-transformed animal cells (Vasiliev et al 1971, Crossin and Carney 1981a, Chou et al 1984 mildew interactions 149 Shinohara et al 1989). Other authors observed that depolymerization of MTs enhanced DNA synthesis in mouse fibroblasts stimulated with peptide growth factors (Friedkin et al 1979(Friedkin et al , 1980.…”

“…In different experimental systems, disorganization of MTs was reported to be a sufficient trigger for mitogenic activation of some quiescent animal cells without the need for otherwise essential growth factors (Chou et al 1984, Miura et al 1987, Tsuji et al 1992. On the other hand, stabilization of MTs with taxol was found to prevent the colchicine-induced enhancement of the G1/S transition (Crossin and Carney 1981b, Chou et al 1984, Shinohara et al 1989, Tsuji et al 1992, as well as the stimulation of DNA synthesis by cytomegalovirus (Ball et al 1990). All these findings support the notion that stable MTs are important for expression of negative control over interphase progression, especially during final stages of the G1 phase, and in this way they seem to participate in execution of the crucial G1/S check-point.…”

Mitotic arrest and toxicity in Biomphalaria glabrata (Mollusca: Pulmonata) exposed to colchicine

“…The increased number of mitotic Wgures at 4-12 h suggests that although colchicine arrested cells at the metaphase stage, it did not block new cells from entering the cell cycle during this time, and possibly could have stimulated mitosis. Although colchicine is a well-known antimitotic agent, in vitro it may stimulate DNA synthesis, and the related microtubule depolymerizing agent colcemid actually promotes cell division (Shinohara et al, 1989).…”

Lysophosphatidic acid and microtubule-destabilizing agents stimulate fibronectin matrix assembly through Rho-dependent actin stress fiber formation and cell contraction.

“…At 10 and 20 mM, there were less abundant stress fibers present after stimulation with LPA or nocodazole, while 40 are similar and mediated by Rho-dependent actin stress fiber formation and cell contraction. The depolymerization of cellular microtubular system elucidates diverse pleiotropic effects on cells, including the assembly of prominent actin cables (Danowski, 1989;Kajstura and Bereiter-Hahn, 1993), an increase in the number and size of vinculin-containing focal adhesions (Lloyd et al, 1977), local tyrosine phosphorylation of the focal adhesion proteins FAK and paxillin (Bershadsky et al, 1996), and the stimulation of DNA synthesis (Crossin and Carney, 1981;Shinohara et al, 1989). The impairment of microtubules may block intracellular vesicular transport and affect signaling by blocking receptor internalization or recycling (Kapeller et al, 1993).…”