Imbalance of total cellular nucleotide pools and mechanism of the colchicine-induced cell activation.

Abstract

Treatment with colchicine or vinblastine, both inhibitors of microtubule assembly, renders quiescent 3T3 cells in an "activated state" as evidenced by induction of DNA synthesis and other criteria. Microtubule disassembly caused by colchicine or vinblastine brings about a dramatic expansion of total cellular UTP pools with a concomitant diminution in total cellular ATP pools, thus resulting in a marked imbalance in total cellular nucleotide pools. Colchicine and vinblastine also stimulate total cellular RNA synthesis without enhancing uridine phosphorylation, suggesting that these drugs affect the G, phase of the cell cycle at a point beyond the enhancement of uridine phosphorylation that usually accompanies mitogenic stimulation of quiescent mammalian cells.The markedly expanded cellular UTP pools appear to be necessary for initiation of the colchicine-stimulated DNA synthesis because decreasing cellular UTP pools by addition of D-glucosamine results in a selective inhibition of DNA synthesis in the colchicine-stimulated, but not control, cells. Furthermore, Dglucosamine exerts its inhibitory effect only when it is present in the cultures within the first 14 hr after colchicine treatment. When added at 21 hr, D-glucosamine still decreases cellular UTP pools, but it is no longer inhibitory for DNA synthesis, which commences 14-16 hr after colchicine stimulation. Taxol, an antitumor drug, prevents microtubule disassembly and also blocks such events as expansion of total cellular UTP pools and stimulation of RNA and DNA synthesis, indicating that microtubule depolymerization acts as a primary event initiating the process of cell activation induced by colchicine.suggest that MT depolymerization is involved in modulating the initiation of DNA replication after mitogenic stimulation, although the underlying mechanism is not known.Since MT assembly in vitro is accompanied by GTP hydrolysis (12, 13), we have studied the effect of the colchicine-stimulated GO/G1 to S phase transition of 3T3 cells on the levels of cellular acid-soluble nucleotide pools, which have been shown to possess a regulatory role in the progression of mammalian cells along their cycle (14-17). We report here that MT disassembly induced by colchicine or vinblastine results in a sharp expansion of total cellular UTP pools with a concomitant diminution in total ATP pools and thus creates an imbalance in the cellular nucleotide pools as evidenced by large increases in the UTP/ATP ratio. In addition, we report that total cellular RNA synthesis, but not uridine phosphorylation, is stimulated following colchicine treatment. Furthermore, we also report that D-glucosamine effectively decreases the expanded cellular UTP pools and causes a selective inhibition of DNA synthesis in colchicinetreated, but not control, cells. Finally, we also report that taxol treatment effectively blocks events that normally occur following MT disassembly induced by colchicine or vinblastine. Taken together, our results suggest that the markedly increased cellular UTP p...

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.…”

“…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).…”

“…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.…”

Cytoskeletal Active Drugs Modulate Signal Transduction in the Protein Kinase C Pathway.

“…Someattempts have been made to decipher the specific biochemical and molecular events involved in mediating the influence of the cytoskeleton on gene expression and cell proliferation (7,15,46,57). To define the role of the cytoskeleton in mitogenic signal transduction in the PKCpathway we have studied the effect of cytoskeletal active drugs on TPA-stimulated mitogenesis in mouse 3T3-L1 cells at both a biochemical and a molecular level.…”

Intracellular nucleotide pools and ratios as tools for monitoring dedifferentiation of primary porcine hepatocytes in culture

“…Additionally, fluctuations in pool size can produce alterations in transport processes, macromolecular synthesis and cell growth. Some evidence has been reported, that the pool size of ATP, the adenylate energy charge (AEC) or the pool size of UTP, influence or correlate with the cell cycle (Rapaport et al 1979), react to the stimulation of cells by serum (Grummt et al 1977) or colchicines (Chou et al 1984) and are involved in growth control (Murphree et al 1974).…”