Cell Cycle-dependent Changes Research Articles

2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNPase) activity in cultured nerve cell lines from central nervous system: comparison of proliferating and resting growth states and cell cycle-dependent activity changes.

1. The present communication is concerned with the expression and cell cycle-dependent regulation of the enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in cultured nerve cell lines derived from the rat central nervous system (CNS). 2. The enzyme activity was measured in relation to two reversible serum-controlled growth states (exponentially growing/quiescent) including a comparison of the enzyme activities in cell lines of neuronal and glial origin as well as in fibroblasts. CNPase is present in all cell types tested, but the enzyme activity is very sensitive to changes in the cellular growth state. Nerve cell lines in exponentially growing cultures express a 3 to 15 times higher specific CNPase activity than the nonneural cell types. In serum-starved quiescent cultures, the differences in specific enzyme activity between the nerve cell lines and the fibroblasts are enlarged even more up to a ratio of about 50 to 150, indicating a specific function of this enzyme within the central nervous system. 3. Neuron-like B104 cells could be stimulated to synchronized growth by serum readdition to quiescent cultures. A series of ordered activity changes of CNPase has been observed after the reinitiation of cell growth. The enzyme is stimulated at two particular stages during the cell cycle, leading to a biphasic activity profile. Maximum stimulation of CNPase correlates with the G1 phase. 4. Hydroxyurea-induced blockage of synchronized B104 cells to traverse the S phase also prevents the subsequent stimulation of CNPase activity. Therefore, we conclude that a correlation exists between the periodic activity changes of CNPase and particular phases of the B104 cell cycle.

Cell cycle specific fluctuations of adenosine 3',5'-monophosphate and prostaglandin binding in synchronized mastocytoma P-815 cells

Endogenous adenosine 3',5'-monophosphate (cAMP) levels in mastocytoma P-815 cells, synchronized either at the G 1/S transition by amethopterin- or double thymidine-block or in mitosis by colcemid block, were highest during late S and early G 2 phases and lowest during mitosis. These cell cycle-dependent changes in cAMP levels were largely accounted for by the changes in adenylate cyclase and phosphodiesterase activities. Similar fluctuations occurred simultaneously with specific prostaglandin E 1 (PGE 1) binding, histidine decarboxylase activity, histamine content, and [ 35S]SO 4 2− incorporation into glycosaminoglycans of the cells. In addition, endogenous levels of the E group of prostaglandins (PGEs) and [ 14C]arachidonic acid incorporations into PGE, phosphatidylcholine and phosphatidylinositol also exhibited fluctuation patterns similar to that of cAMP levels. Since cAMP levels still fluctuated in a serum-depleted medium where DNA synthesis and cell division were inhibited, endogeneous levels of prostaglandin and cAMP appeared not to be regulated solely by serum factor(s). Exposure of cells at G 1/S transition to 1-methyl-3-isobutylxanthine (MIX) resulted in a 10-fold elevation of cAMP levels throughout the cell cycle without affecting DNA synthesis. On the other hand, PGE 1 and/or MIX added at late S phase elevated cAMP levels, prolonged G 2 phase and retarded the cell division, but these agents added at the beginning of mitosis elevated cAMP levels without affecting the cell division. These results suggest that prostaglandins newly synthesized by the increased metabolism of phospholipids promote the cAMP synthesis via their binding to the receptors and thereby control the division and phenotypic expression of mastocytoma P-815 cells.

Glucocorticoids and lymphocytes. II. Cell cycle-dependent changes in glucocorticoid receptor content.

To study variations in glucocorticoid receptor levels during the cell cycle, we have separated mitogen-stimulated human peripheral lymphocytes and rat lymph node cells by unit gravity sedimentation and measured glucocorticoid binding in the resultant fractions. By morphologic criteria and thymidine incorporation, the fractions were separated into populations of G0 and G1 phase and S and post-S phase cells. A 2- to 3-fold increase in glucocorticoid receptor sites per cell, for cells in the S and post-S phase over those in G0 and G1, was observed with both nonstimulated rat lymph node cell suspensions and concanavalin A-stimulated human peripheral lymphocytes. These observations together with those from other studies indicate that formation of new glucocorticoid receptors near the S phase may be a general phenomenon in proliferating cells. We propose that this increase in glucocorticoid receptors during the cell cycle may explain the increase in glucocorticoid receptors in mitogen-stimulated lymphocytes.

Guinea-pig keratinocytes and melanocytes in tissue culture: scanning, transmission and high voltage electron microscope observations.

Keratinocytes and melanocytes cultured from guinea-pig epidermis were studied with scanning, transmission and high voltage electron microscopy to characterize the surface and internal morphology. Keratinocytes exhibited contact-inhibition and a range of surface structures consistent with cell-cycle dependent changes. Stereoscopic analysis of high voltage electron micrographs indicated regular oval nuclei with nucleoli at different depths, while thin sections revealed local channels in the nuclei. Secondary cultures differed from primary cultures in the disorder of the microfilaments, in the failure to form desmosomes, and in the failure of melanocytes to persist in culture. The beaded surface of melanocytes was indicative of underlying melanosomes that were seen in high voltage micrographs. Melanocytes were rounded with moderate ruffles or were dendritic with ruffles on the termini. These findings are discussed in relation to the observational techniques and in relation to modes of locomotion of and pigment transfer to epidermal cells.

Comparison of transcription stimulating, phenol-soluble non-histone chromosomal proteins in normal rat liver and transplantable hepatocellular carcinomas

The non-histone chromosomal proteins (NHCP) of a rapidly and slowly proliferating transplantable hepatocellular carcinoma (THC) were compared to those of normal and regenerating rat liver. The total quantity of NHCP is approximately threefold higher in the THCs than in either normal rat liver at 4 h and 44 h regenerating rat liver. Only those NHCP that can be extracted from chromatin by 0.35 M NaCl were further examined and it was observed that the proteins of this highly complex fraction could be further fractionated by their differential phenol-solubility. The phenol-soluble 0.35 NHCP contained protein(s) capable of stimulating the level of DNA-directed RNA synthesis in vitro. The total amount of this stimulatory activity was 5 times higher in the rapidly growing THC and 1.6 times higher in the slowly growing THC than in normal rat liver. In order to assess the contribution of cell-cycle dependent alterations on the increase in the amount of stimulatory activity in the THCs, 44 h regenerating rat livers were examined. This tissue represents a mix of cells in various stages of the cell cycle which is similar to that found in the THCs. It was found that the total quantity of NHCP in the 44 h regenerating rat liver was the same as in normal rat liver. The total amount of the stimulatory activity also was similar in both the normal and 44 h regenerating rat liver. The amount of the stimulatory activity was found to double in 4 h regenerating rat liver, however. These data suggest that the alterations observed in the NHCP of the THCs are not due solely to cell cycle dependent changes, but may represent malignancy dependent alterations.

Cell cycle-dependent changes in non-membranous nuclear ghosts from HeLa cells.

ABSTRACT Non-membranous nuclear ghosts are isolated from the nuclei of cultured HeLa cells after successive treatments with detergents and high ionic strength (0·5 M MgCl2) buffers. These ghosts have been shown by Keller & Riley to consist of a network consisting of annuli (90 nm in diameter), rod-like structures and dense bodies interconnected by DNase-sensitive strands. In nuclear ghosts purified from HeLa cells synchronized by a double thymidine block technique, the arrangement of these components exhibits dramatic cell cycle-dependent organizational differences. During G1 the small dense bodies (or aggregates) are widely dispersed and appear to be associated with the surface of the ghost. In S-phase, only a single (large) dense body is present and it is located in the centre of the ghost. The appearance of nuclear ghosts from cells in G2 is intermediate between those observed in G1 and S. These cell cycle-dependent organizational differences in the ultrastructure of nuclear ghosts are paralleled by differences in their susceptibility in suspension to DNase I. The G1 -phase ghost is markedly sensitive, whereas the S-phase ghost has both a sensitive component, the network consisting of annuli and rod-like structures connected by thin strands, and a resistant component, the single (large) dense aggregate. The unique morphology of ghosts at a particular phase of the cell cycle and the similarity of the polypeptide species present in ghosts isolated at these 3 phases of the cell cycle lead us to conclude that the various organizational patterns described are alternative cell cycle-dependent configurations of what is fundamentally the same macromolecular complex. A model is presented in which internal nuclear skeletal components and the non-membranous nuclear envelope are considered to be dynamic interacting components of interphase nuclei. This model provides a physiological rationale for nuclear envelope expansion capability which has been observed under experimental conditions by many investigators.

Cell cycle dependent changes in morphology: Studies with a cold-sensitive mutant of Chinese hamster ovary cells

cs4-D3, a cold-sensitive mutant of CHO cells is defective at the G1 interval of the cell cycle and exhibits a reversible change in morphology from a fibroblast appearance to an “epithelial-like” form. Here, we have shown by time-lapse microcinematography that change in morphology, in either direction, is a post-mitotic event.

Cell cycle dependent changes in potassium transport.

K transport has been investigated during progression of cultured Ehrlich ascites tumor cells through the cell cycle. Using a double thymidine block technique, Ehrlich cells carried in continuous culture have been synchronized, as verified by simultaneous monitoring of cell number, cell volume, 3H-thymidine incorporation and mitotic index. Unidirectional influx, efflux and cell content of K have been monitored throughout the cell cycle. The nature of the pump mediated, ouabain-sensitive K flux and the furosemide-sensitive component of K flux, presumably representing K-K exchange, have also been evaluated. In early S period the ouabain sensitive component, representing the Na-K pump, comprises 52% of the total unidirectional K influx and subsequently declines during G2 period to a minimum of 40% in mid G2. During M and early S the activity again rises. As the ouabain sensitive component becomes maximal in late S period, the furosemide sensitive component diminishes from approximately 30% of the total influx to approximately 10%. The same pattern is observed in the G2 period. As the pump component diminishes, the furosemide sensitive component increases. Furosemide sensitive K efflux has also been monitored and the pattern is equivalent to that observed in the invlux studies. No change in net K flux is observed in the presence of furosemide. This indicates that the furosemide sensitive component represents an exchange component for K. These results are consistent with the conclusion that the alterations in exchange and pump fluxes are physiological events associated with progression of the cell cycle.

HeLa cell plasma membranes: Changes in membrane protein composition during the cell cycle

HeLa cells grown in suspension culture were synchronized by amethopterin block and thymidine reversal. In some cases an additional Colcemid block was used to obtain mitotic cells. From the various phases of the cell cycle, cells were harvested and the plasma membranes isolated. The membrane proteins were solubilized in sodium dodecyl sulphate and separated by gel electrophoresis in the presence of sodium dodecyl sarcosinate. About 35 protein bands, five of which were stained with periodic acid-Schiff reagent, appeared. Most of the bands were identical in all membrane preparations, but a few minor bands seemed to be associated with limited periods of the cell cycle. In particular, the cells in mitosis apparently contained plasma membrane proteins which did not occur in other phases. Amino acid analyses of the plasma membranes revealed no significant cell cycle-dependent changes in the amino acid composition.

Cell surface changes and Rous sarcoma virus gene expression in synchronized cells.

We have investigated whether cell surface changes associated with growth control and malignant transformation are linked to the cell cycle. Chicken embryo cells synchronized by double thymidine block were examined for cell-cycle-dependent alterations in membrane function (measured by transport of 2-deoxyglucose, uridine, thymidine, and mannitol), in cell surface morphology (examined by scanning electron microscopy), and in the ability of tumor virus gene expression to induce a transformation-specific change in membrane function. We reach the following conclusions: (a) The high rate of 2-deoxyglucose transport seen in transformed cells and the low rates of 2-deoxyglucose and uridine transport characteristic of density-inhibited cells do not occur in normal growing cells as they traverse the cell cycle. (b) Although there are cell cycle-dependent changes in surface morphology, they are not reflected in corresponding changes in membrane function. (c) Tumor virus gene expression can alter cell membrane function at any stage in the cell cycle and without progression through the cell cycle.

Histones from exponential and stationary L-cells: Evidence for metabolic heterogeneity of histone fractions retained after isolation of nuclei

Histones of exponential and G 1-arrested mouse L-cells were double-labeled with either [ 3H]lysine and [ 32P]phosphate or with [ 14C]arginine and [ 3H]acetate in order to investigate cell cycle-dependent changes in rates of synthesis and metabolic modifications as well as the effect of the method of nuclear isolation on retention of the labeled fractions. Results indicate that newly synthesized lysine-rich histones incorporate 32P at the highest rate. However, phosphorylation can also be detected in G 1-arrested cells and in the case of F 2b, F 1 0 , and F 1 1 , with no detectable new synthesis. On the other hand, acetylation proceeded at similar rates in both exponential and stationary cells with the exception of F 2a 2 and F 3 which showed higher acetylation levels in the latter. In relation to the method used for nuclear isolation, we observed that, even in cases where no difference in the relative amount of a given histone could be detected, the species recovered were not identical. We conclude that none of the methods commonly employed retains all of the histones. In general, the acetylated species appear to be best preserved at a higher divalent cation concentration while the newly synthesized ones are better conserved at lower ionic strengths.

Histones from exponential and stationary L-cells: Evidence for differential binding of lysine-rich and arginine-rich fractions in chromatin

Histones from exponential and stationary-phase mouse L-cells were quantitated after acrylamide gel electrophoresis in order to investigate cell cycle-dependent changes in the mode of binding of the various fractions in chromatin. By introducing various concentrations of citrate and divalent cations in the medium used for cell lysis and isolation of nuclei prior to histone extraction it was possible to demonstrate that certain histone fractions are preferentially retained in either exponential or stationary-phase nuclei. Differential retention of lysine-rich F 1 was most evident when the lysing medium contained 1 m m Mg 2+ and Ca 2+ and 5 m m citrate (pH 2.75). In these conditions twice as much F 1 is retained in stationary as in exponential nuclei. Differential retention of arginine-rich histones was most evident when the lysing medium contained 10 m m Mg 2+ and Ca 2+ and no citrate. In these conditions more F 2a 1 is retained in exponential than in stationary nuclei while the opposite is true for F 3. However, the total amount of arginine-rich fractions (F 2a 1 + F 3) retained was found to be the same in both cell phases. The results are discussed in relation to known structural features of the histones.

Cell-cycle dependent changes in sensitivity to γ-rays in synchronously dividing yeast culture

Changes in survival of yeast cells following γ-irradiation at different stages of the cell cycle were studied using a well synchronized culture. Maximum radioresistance occurs at the end of the S phase. Maximum radiosensitivity is observed just before entry into the S phase. The high degree of synchrony obtained allows more precise measurement of the extent of survival changes than has been achieved until now with partially synchronized cultures. Indeed, after a 60 krad irradiation we find a 100 % survival for cells which have just finished the S phase of the first cell cycle, against a 2 % survival for cells which are ready to enter the S phase of the second cell cycle. As the culture desynchronizes through successive cell cycles we have been able to follow the way in which survival curves are modified. We can extrapolate that with a perfectly synchronized culture the survival of ‘early S’ cells to a 60 krad irradiation would not be 2 % but 0.01 %. The high radioresistance observed at the end of S phase can hardly be explained simply in terms of DNA target or accumulation of radioprotectors. More likely the end of the S phase is a favourable stage for repair processes, at which time two genomes are able to recombine.