demecolcine and Liver-Neoplasms

Intracellular and extracellular levels of 3':5'-cyclic GMP and 3':5'-cyclic AMP were studied in synchronized Novikoff rat hepatoma cells. Intracellular levels of cyclic GMP increased spontaneously from 2-fold (without colcemid) to 10-fold (with colcemid), in proportion to the number of cells in mitosis. As cells entered mitosis, cellular cyclic AMP declined simultaneously with the rise in cyclic GMP. These reciprocal changes in cyclic nucleotide levels were reversed as cells passed out of metaphase and through anaphase. Maximum cyclic AMP and minimum cyclic GMP concentrations occurred during G-1. Less marked reciprocal fluctuations in both cyclic nucleotides were also found in S-phase and early G-2, where the ratio of cyclic AMP to cyclic GMP concentrations first fell and then increased. These changes in cyclic nucleotide ratios were closely correlated with major cell-cycle transitions at the boundaries between G-1/S-phase, S-phase/G-2, G-2/prophase, and metaphase/anaphase. Most, but not all, of the extracellular cyclic nucleotides were extruded when cells traversed mitosis. Colcemid or vinblastine completely prevented the appearance of extracellular cyclic AMP but augmented the appearance of extracellular cyclic GMP in parallel with the accumulation of mitotic cells. These results reflected changes in intracellular cyclic nucleotides and indicated that increased intracellular turnover of cyclic GMP and cyclic AMP occurred before and after metaphase, respectively. Elevated cyclic GMP levels during mitosis and S-phase are consistent with potential modulatory roles for this cyclic nucleotide in proliferation.

Topics: Bleomycin; Carcinoma, Hepatocellular; Cell Division; Cell Line; Cyclic AMP; Cyclic GMP; Demecolcine; Liver Neoplasms; Mitosis; Neoplasms, Experimental; Vinblastine

Topics: Biological Transport; Carcinoma, Hepatocellular; Cell Division; Cell Line; Demecolcine; DNA Replication; Endocytosis; Horseradish Peroxidase; Liver Neoplasms; Mitosis; Peroxidases

Populations of Novikoff rat hepatoma cells (subline N1S1-67) were monitored for the rates of transport of various substrates and for their incorporation into acid-insoluble material as a function of the age of cultures of randomly growing cells in suspension as well as during traverse of the cells through the cell cycle. Populations of cells were synchronized by a double hydroxyurea block or by successive treatment with hydroxyurea and Colcemid. Kinetic analyses showed that changes in transport rates related to the age of cultures or the cell cycle stage reflecte alterations in the V max of the transport processes, whereas the Km remained constant, indicating that changes in transport rates reflect alterations in the number of functional transport sites. The transport sites for uridine and 2-deoxy-D-glucose increased continuously during traverse of the cells through the cell cycle, whereas those for choline and hypoxanthine were formed early in the cell cycle. Increases in thymidine transport sites were confined to the S phase. Synchronized cells deprived of serum failed to exhibit normal increases in transport sites, although the cells divided normally at the end of the cell cycle. Arrest of the cells in mitosis by treatment with Colcemid prevented any further increases in transport rates. The formation of functional transport sites was also dependent on de novo synthesis of RNA and protein. Inhibition of DNA synthesis in early S phase inhibited the increase in thymidine transport rates which normally occurs during the S phase, but had no effect on the formation of the other transport systems. Transport rates also fluctuated markedly with the age of the cultures of randomly growing cells, reaching maximum levels in the mid-exponential phase of growth. The transport systems for thymidine and uridine were rapidly lost upon inhibition of protein and RNA synthesis, and thus seem to be metabolically unstable, whereas the transport systems for choline and 2-deoxy-D-glucose were stable under the same conditions.

Topics: Animals; Biological Transport; Carcinoma, Hepatocellular; Cell Division; Cells, Cultured; Choline; Demecolcine; Deoxyglucose; DNA; Glucose; Hydroxyurea; Hypoxanthines; Kinetics; Liver Neoplasms; Mitosis; Neoplasms, Experimental; Nucleosides; Rats; Receptors, Drug; RNA, Neoplasm; Spectrometry, Fluorescence; Thymidine; Time Factors; Tritium; Uridine

The phosphorylating of the lysine-rich histone at various stages in the cell cycle has been studied. In rapidly dividing cell populations the lysine-rich histone is phosphorylated rapidly after synthesis and more slowly once bound to the chromosome. The half-life of hydrolysis of such interphase phosphorylation in 5 hr except during mitosis when the phosphata hydrolysis increases almost three-fold. During mitosis there is extensive phosphorylation at sites different from those phosphorylated during interphase and a smaller measure of sites common to both mitotic and interphase cells. The sites of mitotic phosphorylation are most critically distinguished from those phosphorylated in interphase by the rapidly hydrolysis of M-phase phosphohistone when the cells divide and enter the G1 phase of the cell cycle.

Topics: Autoradiography; Carcinoma, Hepatocellular; Cells, Cultured; Demecolcine; DNA, Neoplasm; Electrophoresis; Histones; Hydroxyurea; Liver Neoplasms; Lysine; Mitosis; Neoplasms, Experimental; Phosphates; Phosphoproteins; Phosphoric Monoester Hydrolases; Phosphorus Radioisotopes; Tritium

The regulation of tyrosine aminotransferase (TAT) activity has been examined in two liver-derived heteroploid cell lines. One (hepatoma tissue culture cells [HTC]) was derived from a hepatoma, the other (rat liver culture cells [RLC]) was derived from normal liver. The two cell lines show the following striking similarities in the control of this specific protein: (a) The kinetics of TAT induction by dexamethasone phosphate (DxP) are similar in randomly growing cells of both lines; (b) During mitosis and early G(1) phase of the cell cycle TAT activity cannot be induced by DxP in either cell line; (c) 2-3 h into G(1), when both lines become sensitive to inducer, basal enzyme activity declines to a new steady-state level; (d) Preinduced cells collected in mitosis show approximately twice the level of TAT activity as fully induced, randomly growing cultures and this activity is maintained in early G(1) with or without the inducer; and (e) Inhibition of RNA synthesis by 5 microg/ml of actinomycin D in preinduced, synchronized cells allows TAT activity to remain at constitutive levels throughout G(1), even in the absence of inducer. These results are presented in support of a previously described model which states that glucocorticoid hormones exert posttranscriptional control of the synthesis of specific proteins in mammalian cells.

Topics: Animals; Carcinoma, Hepatocellular; Cell Division; Cell Line; Dactinomycin; Demecolcine; Dexamethasone; Enzyme Induction; Kinetics; Liver; Liver Neoplasms; Mitosis; Neoplasms, Experimental; Rats; Time Factors; Tyrosine Transaminase